Steroide anionic compounds, method of their production, usage and pharmaceutical preparation involving them

ABSTRACT

A compound with general formula I 
     
       
         
         
             
             
         
       
         
         
           
             for treatment of various diseases of the central nervous system, in treatment of neuropsychiatric disorders related to imbalance of glutamatergic neurotransmitter system, ischemic damage of CNS, neurodegenerative changes and disorders of CNS, affective disorders, depression, PTSD and other diseases related to stress, anxiety, schizophrenia and psychotic disorders, pain, addictions, multiple sclerosis, epilepsy and gliomas.

FIELD OF THE INVENTION

This invention is represented by, anionic steroid compounds, ways oftheir production, their applications and pharmaceutical substancescontaining them. The invention particularly deals with pregnanolonederivatives substituted in 3alpha-position with the anionic group boundin this position. These derivatives may be beneficial in treatment ofseveral central nervous system (CNS) diseases, especially ischemic CNSinjury, neurodegenerative alterations and diseases, depression,post-traumatic stress disorder and other stress-related disorders,schizophrenia and various psychotic diseases, pain, addiction, multiplesclerosis and autoimmune disorders, epilepsy, and gliomas as well asother CNS tumors.

BACKGROUND ART

Glutamate is the principal excitatory neurotransmitter in the centralnervous system of mammals. During synaptic transmission, thepost-synaptic responses occur via ionotropic and metabotropic glutamatereceptors. Metabotropic receptors operate via G-proteins and mobilizecalcium ions from intracellular compartments. Activation of ionotropicreceptors results in increase in permeability of postsynaptic membranefor sodium, potassium and calcium cations by opening a ion channel,which is an integral parts of the receptors.

Typical examples of ionotropic receptors are N-methyl D-aspartate (NMDA)receptors, AMPA and kainate receptors. Although current knowledgesuggests specific role of various types of superfamily of glutamatereceptors in the glutamate-induced excitotoxicity, ionotropic receptorsare generally considered to be a key player in these processes.Activation of ionotropic receptors leads to alterations in intracellularconcentrations, of various ions, mainly of Na⁺ and Ca²⁺. Currentresearch demonstrates that beside calcium, elevated intracellular levelsof sodium ions can also lead to neuronal death. In neuronal cultures andin retina the activation of glutamate receptors may lead to damage evenby sodium cations in absence of extracellular calcium ions. Nonetheless,toxicity of elevated glutamate levels is usually associated withelevations in intracellular concentrations of Ca²⁺. Currently it is wellestablished that there is a direct relationship between excessive influxof calcium into cells and glutamate-induced damage to neurons.Glutamate-induced pathological calcium elevation is usually ascribed toprolonged activation of ionotropic receptors. Elevation in intracellularcalcium then may trigger the down-stream neurotoxicity cascade, whichinvolves uncoupling of mitochondrial electron transport from ATPproduction, supernormal activation of enzymes such as calpain and otherproteases, induction of specific protein kinases, NO-synthase,calcineurins and endonucleases. These changes may also promote theproduction of toxic reactive molecules such as reactive oxygen species(ROS) and induce changes in cytoskeleton architecture and activation ofsignals leading to apoptosis and mitochondrial damage (Villmann andBecker, 2007).

A number of preclinical studies show a remarkable ability of NMDAreceptor antagonists to prevent from the excessive exocytose ofglutamate and damage to the CNS. From the clinical point of view;however, their therapeutic potential is rather limited. Regarding thefact that glutamate receptors are ones of the most abundant in the CNS,application of their antagonists leads to wide variety of side effects,ranging from motor impairment to induction of psychotic symptoms. On thecontrary, a large divergence of NMDA receptors and differences in theirdistribution at synapses and at extrasynaptic sites offer a possibilityto search for drugs which selectively influence only a limited subset ofNMDA receptors and thus to avoid the induction of unexpected sideeffects, while retaining their therapeutic neuroprotective activity.

Previous results demonstrated that naturally occurring 3α5β-pregnanolonesulfate affects the activity of NMDA receptor by a use-dependent manner.As a consequence this molecule has a more pronounced inhibitory actionon the tonically active NMDA receptors than on those phasicallyactivated by glutamate during synaptic transmission. It was alsodemonstrated that activation of extrasynaptic tonically activated NMDAreceptors is very important for excitotoxic action of glutamate(Petrovic et al., 2005).

Therefore, we have started the development and testing of novel NMDAreceptor antagonists derived from neurosteroids. These newly synthesizeddrugs exhibit affinity for extrasynaptic NMDA receptors. What is moreimportant, previous electrophysiological studies showed that thesecompounds bound preferentially to open NMDA receptor channels. Ourcompounds lack affinity for other types of receptor; it is thus presumedthat they will not affect signal transmission between neurons. Thesuggested mechanisms of their action are the blockade of extrasynaptictonically activated NMDA receptors and prevention of excessive action ofglutamate on neurons.

In the last decade, the biomedical research focused on the study of therole of neurosteroids in the pathogenesis of number of neuropsychiatricdiseases and evaluation of their therapeutic potential. Mechanisms ofaction of neurosteroids are conventionally associated with theiractivity on NMDA and GABA-A receptors. A number of experimental studieswith animal models show their potential in therapy of several diseasesof CNS, including neurodegenerative disorders, multiple sclerosis,affective disorders, alcoholism, pain, insomnia or schizophrenia(Morrow, 2007; Weaver, 2000).

Neurosteroids also play a crucial role in the regulation of reactivityto stress and stress-related CNS disorders. Corticosteroid levels areknown to acutely increase after exposition to a stressor; thisrepresents an adaptive mechanism. On the other hand, experimental modelsof chronic stress and depression in laboratory rodents show decreasedlevels of neurosteroids both in brain and plasma. Similar findings areoften reported in patients suffering from depressions andpre-menstruation syndrome suggesting impairments in the CNS homeostaticmechanisms in stress-related neuropsychiatric disorders.

Steroid compounds affect activity and plasticity of neural and glialcells during early in life, and later in development they play anessential trophic and neuroprotective role in the adult CNS. Steroidsare released by sexual and adrenal glands as well as in the CNS.Steroids secreted by peripheral glands reach brain, medulla and spinalcord via blood circulation. Nonetheless, some neural steroids (i.e.,neurosteroids) are synthesized directly in the CNS. The most studiedneurosteroids are represented by pregnenolone, progesterone,dehydroepiandrosterone (DHEA) and their reduced metabolites and sulphateesters. Not much is known about regulation of neurosteroid synthesis inthe CNS, but it is generally assumed that they may underlie interactionof multiple cell types in the CNS. For example, synthesis ofprogesterone by Schwann cells surrounding peripheral nerves is regulatedby signals diffusing from neurons.

Neurotrophic and neuroprotective properties of some neurosteroids wereconvincingly demonstrated both in cultures and in vivo. Progesteroneplays a pivotal role in neurological recovery from traumatic brain andspinal cod injury by mechanisms including protection against excitotoxicdamage to the brain, lipid peroxidation and by induction expression ofspecific enzymes. For example, after cutting the spinal cord, thissteroid increases the number of NO-synthase-expressing astrocytes inplace adjacent to cut both in the distal and proximal segment of thecord.

This steroid was also shown to regulate formation of new myelin sheaths.This fact was shown in regenerating rat sciatic nerve in the culturewith sensory neurons and Schwann cells. Progesterone also supportsmyelination by activation of genes coding for proteins participating inthis process.

As mentioned before, neurosteroids importantly modulate the function ofmembrane receptors for various neurotransmitters, namely GABA_(A)receptors, NMDA receptors and sigmal-opioid receptors. These mechanismsare most likely responsible for psychopharmacological effects ofsteroids and may at least partly account for their anticonvulsant,anxiolytic, neuroprotective and sedation effects as well as for theirinfluence upon learning and memory functions. For instance, pregnanolonesulphate was shown to be capable of reversing cognitive deficit in agedanimals and exerting a protective effect on memory in several amnesiamodels. Recent studies have demonstrated direct effect of neurosteroidson intracellular receptors. Despite absence of direct evidence forbinding of neurosteroids to corticoid receptors, they may obviouslymodulate their function indirectly, by interaction with protein kinasesC and A, MAP-kinase (MAPK) or CaMKII. Moreover, pregnanolone andpregnanolone sulphate were shown to affect microtubule-associatedproteins and increase the rate of microtubule polymeration, which may inturn affect neuronal plasticity. We are far from fully understandingthese newly-described effects of neurosteroids, however, their potentialrole in neuroprotective mechanisms deserves scientific attention.

Sulfated esters of neurosteroids also play a physiological role in theregulation of receptors for excitatory and inhibitory neurotransmittersand participate in the natural protective properties of CNS tissue.Sulphated esters of neurosteroids and their analogues are promisingmolecules, potentially beneficial for treatment of CNS disorders.Nonetheless, a ratio between neurosteroids and their sulfated esters ismaintained enzymatically in the CNS tissue in vivo. Exogenousadministration of sulfated esters may not lead to improvement in theprotective functions due to increased enzyme activity in the CNSconverting them to inactive forms. The invented molecules aremetabolically stable analogues of sulfated esters of neurosteroids;moreover, they pass the blood-brain barrier more readily due to theirchemical structure. Sulfated and thus polar steroids compounds generallypenetrate the blood-brain barrier with difficulty, but it wasdemonstrated that intravenously administered pregnanolone sulphate canreach the brain. This transport of sulphated analogs is probablymediated by active exchange mechanisms associated with so-called organicanion transport protein (OATP), which is expressed in the cellsthroughout the CNS.

Advantage of our molecules is that they retain similar pharmacologicaland physiological properties as pregnanolone sulphate, but they are notdegraded by sulfatases into non-conjugated metabolites.

DESCRIPTION OF THE INVENTION

The present invention relates to compounds of general formula I

in which

-   -   R¹ represents the group of general formula R³OOC—R²—C(R⁴)—R⁵,        where R² means alkyl or alkenyl group with 1 to 18 carbon atoms        in a straight or a branching carbon chain, which may be        substituted by one or more halogen atoms and amino group, which        may be either free or protected by a removable protecting group,        alkoxycarbonyl group, aromatic group, and/or heterocyclic group,        in which the heteroatom means oxygen atom, sulfur, or nitrogen        atom. R³ represents either a hydrogen atom or a protecting group        of carboxyl groups, preferably benzyl group; R⁴ represents        oxygen atom, nitrogen atom, or a sulfur atom bound by a double        bond, or R⁴ represents two hydrogen atoms. R⁵ represents any        minimally bivalent atom, preferably an oxygen atom, the        nitrogen, or carbon atom, except when R² represents the group        (CH₂)_(n), where n=0-3, and simultaneously R³ represents a        hydrogen atom and R⁴ and R⁵ represents an oxygen atom.

The invention is based on results of our experiments, in which effectsof pregnanolone sulphate on native and recombinant NMDA receptors. Thesestudies have demonstrated that this naturally occurring neurosteroidinhibits the responses to exogenous application of NMDA receptoragonists. We have demonstrated that pregnanolone sulphate boundexclusively to activated NMDA receptors (i.e., use-dependent action),but it did not bind to the ionic pore of the receptors, as did othersubstances as Mg²⁺, ketamine, dizocilpine or memantine. Binding kineticsand mechanism of action of pregnanolone sulphate may result topreferential increase in inhibitory action on tonically-active glutamatereceptors rather than phasically-activated receptors involved in fastsynaptic transmission. The newly synthesizes analogues, which aresubject to this invention, have the same mechanism of action on the NMDAreceptors as pregnanolone sulphate.

Moreover, since exogenous administration of pregnanolone sulphate doesnot often lead to beneficial effects due to increase enzymatic activityof sulphatases, our molecules are their non-hydrolysable analogues.

Endogenous 3α-C sulphated neurosteroids have therapeutic potential, buttheir clinical use is complicated due to their metabolic andpharmacokinetic properties. First, these neurosteroids are metabolicallyconverted through the action of steroid sulfatase to drugs with oppositebiological effect. Second, they do not easily cross the blood-brainbarrier (BBB). Third, they have side effects originating in their NMDAreceptor antagonism.

Presented compounds are not converted by enzymes, they cross the BBB,and they do not show the side effects of NMDA antagonism. Furthermore,they show potentially therapeutic effect in animal models of CNSdisorders.

This invention relates also to the method of production of abovementioned compounds of general formula I where R¹ is as indicated above.The method of production of compound of general formula I, where R¹means the same as above and R⁵ represents oxygen atom, starts from3alfa-hydroxy-5beta-pregnan-20-one of formula II

This compound of formula II can be transferred to the compound ofgeneral formula I, where R¹ means the same as above and R⁵ representsoxygen atom as follows: the particular dicarboxylic acid, dicarboxylicacid with protected amino group or, where applicable, dicarboxylic acidprotected on a one carboxylic group, is dissolved in a suitable solventthat allows to remove the remaining water, preferably in benzene ortoluene, most preferably in benzene. Whereupon after the removal ofwater by a partial distillation of the solvent, the reaction mixturewhich is prevented against the water supply in an appropriate mannerknown in the scope of technique, cooled down to room temperature andunder the inert atmosphere is slowly added condensing agent, preferablyDCC, and a solution of compound formula II in a suitable solvent,preferably in an aromatic hydrocarbon, advantageously in benzene ortoluene, most preferably in benzene, in the presence of a catalyticagent, preferably DMAP. This reaction mixture is stirred 10-48 hours,preferably overnight, at temperatures from 0 to 50° C., preferably atroom temperature. The next day the mixture is poured into saturatedsodium bicarbonate, preferably aqueous NaHCO₃ or KHCO₃, and the productis extracted with an organic solvent, in which is well soluble, forexample, with advantage, ethyl acetate. Collected organic phases arewashed with water to remove sodium bicarbonate. PrecipitatedN,N′-dicyclohexylurea is filtered off and the filtrate is dried overdrying agent, preferably magnesium sulfate or sodium sulfate, mostpreferably sodium sulfate and the solvent evaporated, preferably undervacuum. The obtained product is purified, where appropriate, with theadvantage by a chromatography on a column of silica gel to afford thecompound of general formula I, where R¹ represents the group of thegeneral formula R³OOC—R²—CO— and R² represents alkyl or alkenyl groupwith 1 to 18 carbon atoms in a straight or a branching carbon chain,which may be substituted by one or several halogen atoms and by aminogroup, which is protected by a group that allows deprotection. R³represents a protecting group for carboxyl groups, preferably benzylgroup.

In case that R³ means benzyl protecting group in compound of formula Irequired removing of this protecting group is realized so that theobtained compound is dissolved in a suitable solvent, preferablyalcohol, most preferably in methanol, and to this solution ahydrogenation catalyst is added, preferably Pd/CaCO₃. After thehydrogenation, the catalyst is filtered off and the solvent isevaporated to afford the product of general formula I in which R¹represents the group of general formula R³OOC—R²—CO—, where R² meansalkyl or alkenyl group with 1 to 18 carbon atoms in a straight or abranching carbon chain, which may be substituted by one or severalhalogen atom and amino group, which is protected by a group that allowsdeprotection. R³ represents a hydrogen atom.

When the compound of general formula I was obtained in which R¹represents the group of general formula R³OOC—R²—CO—, R² representsalkyl or alkenyl group with 1 to 18 carbon atoms in a straight or abranching carbon chain which may be substituted by one or more halogenatoms and amino group, which is protected by a removable group, and R³represents a hydrogen atom and has an amino group, which is protected bya removable group, the deprotection of amino groups is accomplished inthe next step so that the compound is dissolved in an organic solvent,preferably in methylene chloride and trifluoroacetic acid is added.Then, the reaction mixture is allowed to react from 0.1 to 48 hours,preferably 16 hours at temperatures from 0° to 50° C., preferably atroom temperature. When the solvent is removed, the residue is dissolvedin an organic solvent, preferably in methanol, then pyridine is addedand the mixture is evaporated to dryness to obtain the product ofgeneral formula I, where R¹ represents the group of general formulaR³OOC—R²—CO—, where R² means alkyl or alkenyl group with 1 to 18 carbonatoms in a straight or a branching carbon chain, which may besubstituted by one or more halogen atoms and amino group; and R³represents a hydrogen atom.

When R² of compound of the general formula I contains a heterocyclicgroup, as for example in the compound of formula HET;

-   -   such heterocyclic group can be introduced into a molecule of        general formula I, for example, by the reaction of activated        carboxyl group with amino substituent on the alkyl chain R².        Carboxyl group may be functionalized with an activating group        (for example with hydroxybenzotriazole, substituted        hydroxybenzotriazole, HATU group, TATU group and advantageously        TSTU group in the form of succinimidylester). This ester reacts        with the compound of general formula I in which R² means alkyl        group substituted with amino group so the compound of general        formula I is obtained, in which R² represents alkyl group        substituted with amino group that is substituted with the        heterocyclic group of HET.

The person skilled in the art can analogously prepare similar compoundsof general formula I, in which R² means as given above.

Thiocompounds and amides can be prepared by analogous procedures fromthe compounds of general formula I, where R means atom of sulfur (asdescribe for instance Swan, Turnbull, Tetrahedron 22, 1966, p. 231), orif appropriate nitrogen atom (as describe for instance Schmitt J.,Panouse J. J., Hallot A., Pluchet H., Comoy P.: Bull Soc. Chim. France1962, p. 1846).

Another subject of this invention is application of compounds of generalformula I, where R¹ means as described before for production oftherapeutics for treatment of neuropsychiatric disorders related todysbalance of glutamatergic neurotransmitter system, especially ischemicCNS injury, neurodegenerative changes and disorders, mood disorders,depression, post-traumatic stress disorder and other stress-relateddisorders, anxiety, schizophrenia and other psychotic illnesses, pain,addiction, multiple sclerosis, epilepsy and gliomas.

Various structural modifications of our invented compounds of generalformula I have shown only minimal differences in their biologicalactivity; these findings are congruent with previouselectrophysiological results using patch-clamp technique and assessingbinding kinetics of these compounds on the NMDA receptors. Therefore, wehave chosen a representative molecule of pregnanolone glutamate fromexample 9 [next: compound from Example 9, compound of a general formulaI, in which R¹ is —CH(NH₂)—(CH₂)₂—COOH group], which was subjected todetailed examination in relation to its neuroprotective action on thehippocampal lesions (by means of NMDA; compound from Example 9). We havealso studied its effects on behavior after separate application and itscomparison with the model molecule (dizocilpine), which is known toexert neuroprotective properties in certain preclinical configurations,but wealth of evidence from animal models and occasional observations inhumans suggest that it posses pronounced psychotomimetic side-effects.

The compound from example 9 penetrates blood brain barrier and rapidlyenter the brain (T_(max)=60 min, c_(max)=508 ng/whole brain) after i.pinjection (dose 1 mg/kg) and exponentially decreases to a mean of 222ng/whole brain after 2 hours, 128 ng/whole brain after 3 hours, 14ng/whole brain after 24 hours and 0.6 ng/whole brain after 48 hours. Itseems that the compound from example 9 is eliminated by a first-orderprocess and it is not cumulated in brain tissue. Next its c_(max)=675ng/ml in plasma at the time of T_(max)=15 min was detected. Thefollowing pharmacokinetics parameters have been estimated for plasma:K_(e)=0.002593; T_(1/2)=267 min; AUC0_inf_(i.p.)=75348.

Subsequent toxicological study showed absense of any signs of acutetoxicity in laboratory rat of the compound from example 9.

Moreover next studies demonstrated absence of hyperlocomotion, deficitin sensorimotor gating and cognitive deficit, the side effects typicalfor noncompetitive NMDA antagonist.

Examples of biological activities indicate possibilities to blockexcessive effect of glutamate in broad spectrum of in animal models ofCNS disorders. The compound from example 9 showed neuroprotective effectin animal models of brain damage induced by hypoxic/ischemic state andby neurotoxic lesion by bilateral injection of NMDA into the dorsalhippocampus. Further the examples documents slight anxiolytic effect andimproving of cognition deficit in models schizophrenia-like behavior.The compound from example 9 exhibits antidepressant properties inextensive tests of depression and stress too. The examples have provenanalgetic and anticonvulsive properties of compound from example 9 inaddition.

The data confirm capability of NMDA receptor antagonists to prevent theexcessive release of glutamate and subsequent damage of the CNS leadingto deterioration of behavior. From the clinical point of view; however,their therapeutic potential is rather limited. Regarding the fact thattheir application leads to wide variety of side effects, ranging frommotor impairment to induction of psychotic symptoms.

Main advantage of 3αC substituted analogues of pregnanolone,use-dependent NMDA antagonists, constitutes absence of serious sideeffect typical for competitive NMDA antagonists, while retaining theirtherapeutic activities.

Biological Activity of the Invented Compounds on Cell Cultures

5-10-day old hippocampal culture cells of HEK293 cultured cells wereused for electrophysiological investigations with a latency of 16-40 hafter transfection. Whole-cell currents were measured by patch-clampamplifier after capacitance and serial resistance. Steroid-containingsolutions were prepared from fresh solution (20 mM) of steroid dissolvedin dimethyl-sulfoxide (DMSO). Same concentrations of DMSO were used inall extracellular solutions. Control and experimental solutions wereapplied via microprocessor-controlled perfusion system with approx. rateof solution exchange in areas adjacent to cells reaching ˜10 ms.

The results show that synthetic analogues of pregnanolone sulphate havethe same mechanism of action on NMDA receptors as pregnanolone sulphate;however, they differ in their affinity for these receptors.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows current responses produced by application of 1 mM glutamateand the effect of neurosteroid from example 9, co-administered (200 μM)with glutamate. Records were made using patch-clamp apparatus fromindividual cultured HEK293 cells transfected with NR1/NR2B receptors.The inhibition index was calculated according to: (1-a/b).100(%).

FIG. 2 Top panel: Total distance traveled in the open-field session as ameasure of spontaneous locomotor activity (according to example 29) wasnot different between control animals and rats treated with compoundfrom Example 9.

FIG. 2 Bottom panel: Prepulse inhibition of the acoustic startle reflex(according to example 29) was not significantly altered by applicationof the compound from Example 9.

FIG. 3 shows total distance traveled by rats in the AAPA task afterapplication of dizocilpine and compound from Example 9 (according toexample 30). * denotes significant difference compared to controls(p<0.05).

FIG. 4 shows number of entrances into the shock sector in the AAPA taskin daily sessions as a measure of cognitive functions afteradministration of dizocilpine and compound from Example 9 (according toexample 31). * denotes significant difference with respect to controls(p<0.05); statistical differences were estimated solely in the finalsession after the controls had reached the asymptotic level ofperformance.

FIG. 5 shows the maximum time avoided per session in the AAPA task as ameasure of cognitive functions after application of dizocilpine andcompound from Example 9 (according to example 32). * denotes p<0.05 withrespect to controls, statistical differences were evaluated in the lastsession at the asymptotic stage of control rats.

FIG. 6 shows the effect of compound from Example 9 at a dose of 0.01mg/kg on the Number of entrances in subsequent AAPA testing (accordingto example 34). # p<0.05 with respect to NMDA alone, * p<0205 withrespect to controls; ** p<0.01 compared to controls.

FIG. 7 shows the effect of compound from Example 9 at a dose 0.01 mg/kgon the maximum time avoided during AAPA testing (according to example35). # p<0.05 with respect to NMDA alone; * p<0.05 with respect tocontrols, ** p<0.01 with respect to controls.

FIG. 8 shows the effect of the compound from Example 9 at a dose 0.1mg/kg on the number of entrances into shock sector during AAPA testing(according to example^(\) 36); * p<0.05 with respect to controls.

FIG. 9 shows the effect of the compound from Example 9 on the maximumtime avoided during AAPA testing (according to example 37).

FIG. 10 shows the effect of the compound from Example 9 on the totalnumber of entrances into shock sector in the AAPA testing (according toexample 38); # p<0.05 compared to NMDA alone, * p<0.05 compared tocontrols.

FIG. 11 shows the effect of the compound from Example 9 on the maximumtime avoided in the AAPA daily sessions (according to example 39). #p<0.05 with respect to NMDA alone, * p<0.05 with respect to controls.

FIG. 12 shows the time course of compound from example 9 concentrationin plasma of rats (ng/ml) after i.p injection thereof (1 mg/kg)according to Example 41. At the y-axis concentrations of above mentionedcompound in plasma are outlined as ng of the compound contained in 1 mlof plasma. At the x-axis the time is outlined as minutes (hours).

FIG. 13 shows the time course of compound from example 9 level in ratbrain (ng/whole brain) after i.p injection thereof (1 mg/kg) accordingto Example 41. At the y-axis levels of above mentioned compound in brainare outlined as ng of the compound contained in whole brain. At thex-axis the time is outlined as minutes (hours).

FIG. 14 shows the effect of compound from example 9 on the body weightof rats in time according to Example 42. The compound was applied on day1 at a single dose of either 1 mg/kg or 100 mg/kg. At the y-axis levelsbody weight in % of first day average are (symbol -♦- for salinetreatment, symbol -▪- for cyclodextrine treatment, symbol -▴- forcompound from exp. 9 (1 mg/kg) treatment, symbol -x- for the samecompound (100 mg/kg). At the x-axis the time is outlined as days.

FIG. 15 shows the effect of compound from example 9 on the weight ofparticular body organs in rats according to Example 42. At the y-axisweight of particular body organs in grams are outlined. At the x-axisthe particular organs are mentioned and type of treatment used (firstcolumn for saline treatment, second column for cyclodextrine treatment,third and fourth column for compound from example 9 treatment in thedoses of 1 mg/kg of b.w. and 100 mg/kg of b.w. respectively).

FIGS. 16A-D show the growth effect of the compound from example 9 onglioma cells in culture according to Example 43. FIGS. 16A and 1613represent control groups of C6 glioma cell lines administered bycholesterol at doses of 0.1 and 1 μg dissolved in 50 ml ofβ-cyclodextrine solution after 3+1 and 3+3 days of cultivation in vitro(DIV) respectively. FIGS. 16 C and 16D represent groups of C6 gliomacell lines administered by the compound of Example 9 at doses of 0.1 and1 μg respectively dissolved in 50 ml of (3-cyclodextrine solution.

FIG. 17 demonstrates effect of compound from example 9 application indoses of 0.001 mg/kg, 0.01 mg/kg, and 10 mg/kg to rats in elevated plusmaze according to Example 44. At the y-axis the number of entries intoopen arms of maze is mentioned. At the x-axis the group of testedanimals are mentioned; first column represents controls, 2.-4. columnrepresents groups of animals administered by above mentioned compound atthe dose of 0.001, 0.01 and 10 mg/kg of b.w. respectively.

FIG. 18 shows total duration of ultrasonic vocalizations as a model ofanxiety according to Example 45. At the y-axis total time of ultrasonicvocalizations in seconds is outlined. At x-axis first column representsgroup of control animals and second column group of animals injected i.p. with drug from example 9 at dose 10 mg/kg.

FIG. 19 visualizes effect of compound from example 9 on prepulseinhibition (PPI) of the startle response in an animal model ofschizophrenia according to Example 46. At the y-axis % of PPI areoutlined. At the x-axis columns represents group of animals: firstcolumn: control group, second and third column: group of animals treatedby compound from ex. 9 in doses of 0.1 mg/kg of b.w. and 1 mg/kg of b.w.respectively; fourth column: group of animals receiving MK-801 (0.1mg/kg of b.w.), fifth and sixth column: group of animals receivingcompound from ex. 9 (0.1 mg/kg or 1 mg/kg of b.w.) in combination withMK-801 (0.1 mg/kg of b.w.).

FIG. 20 shows the effect of compound from example 9 alone or incombination with MK-801 on the locomotion in the open-field behavioraccording to Example 47. At the y-axis total distance in cm traveledduring 30 min in a box is outlined. At the x-axis columns representsgroup of animals: first column: control group, second and third column:group of animals treated by compound from ex. 9 in doses of 0.1 mg/kg ofb.w. and 1 mg/kg of b.w. respectively; fourth column: group of animalsreceiving MK-801 (0.1 mg/kg of b.w.), fifth and sixth column: group ofanimals receiving compound from ex. 9 (0.1 mg/kg or 1 mg/kg of b.w.resp.) in combination with MK-801 (0.1 mg/kg of b.w.).

FIG. 21 shows the effect of the compound from example 9 on the locomotoractivity in the final session of the 4-day AAPA training according toExample 48. At the y-axis total distance (in meters) travelled in thearena is outlined. At the x-axis columns represents group of animals,from left to right are outlined groups receiving saline, MK 801 (0.1mg/kg of b.w.), MK 801 (0.1 mg/kg)+compound from ex. 9 (0.001 mg/kg), MK801 (0.1 mg/kg)+compound from ex. 9 (0.01 mg/kg), MK 801 (0.1mg/kg)+compound from ex. 9 (0.1 mg/kg), MK 801 (0.1 mg/kg)+compound fromex. 9 (10 mg/kg). ### indicates p<0.001 compared to controls. Statisticdifferences were evaluated in last session where asymptotic level ofperformance is reached by controls.

FIG. 22 shows the effect of the compound from example 9 on thedizocilpine-induced avoidance deficit according to example 49. At they-axis number of entrances to shock sector is outlined. At the x-axiscolumns represents group of animals, from left to right are outlinedgroups receiving saline, MK 801 (0.1 mg/kg of b.w.), MK 801 (0.1mg/kg)+compound from ex. 9 (0.001 mg/kg), MK 801 (0.1 mg/kg)+compoundfrom ex. 9 (0.01 mg/kg), MK 801 (0.1 mg/kg)+compound from ex. 9 (0.1mg/kg), MK 801 (0.1 mg/kg)+compound from ex. 9 (10 mg/kg). * indicatessignificant difference compared to controls (p<0.05), ## p<0.01 and ###p<0.001. Statistic differences were evaluated in last session whereasymptotic level of performance is reached by controls.

FIG. 23 shows the maximum time of avoidance as a measure of cognitivefunctions in the final session of the 4-day AAPA task training accordingto Example 50. At the y-axis maximum time avoided in seconds isoutlined. At the x-axis columns represents group of animals, from leftto right are outlined groups receiving saline, MK 801 (0.1 mg/kg ofb.w.), MK 801 (0.1 mg/kg)+compound from ex. 9 (0.001 mg/kg), MK 801 (0.1mg/kg)+compound from ex. 9 (0.01 mg/kg), MK 801 (0.1 mg/kg)+compoundfrom ex. 9 (0.1 mg/kg), MK 801 (0.1 mg/kg)+compound from ex. 9 (10mg/kg). * indicates significant difference compared to controls p<0.05;# p<0.05 and ### p<0.001 compared to MK-801 group. Statistic differenceswere evaluated in last session where asymptotic level of performance isreached by controls.

FIG. 24 shows the effect of compound from Example 9 on the learnedhelplessness model of affective disorders according to Example 51. Atthe y-axis number of escapes is outlined. At the x-axis first columnrepresents group of control animals, second column group of animals fromlearned helplessness group received no drug and third column group ofanimals from learned helplessness group received compound from ex. 9 (1mg/kg of b.w.). ***indicates p<0.001 compared to control group and ##p<0.01, compared to LH group.

FIG. 25 shows the effect of compound from Example 9 on time toimmobility in Forced swimming test according to Example 52. At they-axis the time to immobility in minutes is outlined. At the x-axisfirst column represents group of control animals, second column group ofanimals receiving compound from ex. 9 (1 mg/kg of b.w.). *** indicatesp<0.001, compared to control group.

FIG. 26 shows the effect of compound from example 9 on depression-likebehavior induced by social defeat in mice according to Example 53. Atthe y-axis the total path in open-field after repeated socialinteraction with an aggressive mouse in arbitrary units is given. At thex-axis first column represents group of control animals, second columngroup of animals receiving compound from ex. 9 (1 mg/kg of b.w.). *indicates p<0.05 compared to controls.

FIG. 27 shows the effect of compound from Example 9 on the pain-inducedlimb reaction according to Example 54. At the y-axis the limbswithdrawal latency in seconds is given. At the x-axis first columnrepresents group of control animals, second and third columns groups ofanimals receiving compound from ex. 9 (1 mg/kg and 10 mg/kg of b.w.resp.) before application of thermal stimulation and after that. **indicates p<0.05 compared to controls, ### p<0.001 compared to compoundfrom ex. 9 in dose 10 mg/kg “before”.

FIG. 28 shows the effect of compound from Example 9 on the pain-inducedtail reaction according to Example 54. At the y-axis the tail withdrawallatency in seconds is given. At the x-axis first column represents groupof control animals, second and third columns groups of animals receivingcompound from ex. 9 (1 mg/kg and 10 mg/kg of b.w. resp.) beforeapplication of thermal stimulation and after that.

indicates p<0.05 compared to controls, ### p<0.001 compared to compoundfrom ex. 9 in dose 10 mg/kg “before”.

FIG. 29A, B shows the effect of compound from example 9 on cognitivecoordination and motor activity according to Example 55.

FIG. 29A demonstrates at the y-axis number of entrances into punishedsector in AAPA. At the x-axis first column represents group of controlanimals, second and third columns groups of ischemic animals withoutmedication and receiving compound from ex. 9 (1 mg/kg of b.w.)respectively. **indicates p<0.01 compared to control rats, ## indicatesp<0.01 compared to ischemic rats.

FIG. 29B demonstrates at the y-axis total path elapsed during a sessionin AAPA (metres). At the x-axis first column represents group of controlanimals, second and third columns groups of ischemic animals withoutmedication and receiving compound from ex. 9 (1 mg/kg of b.w.)respectively.

FIG. 30 represents chart showing the effect of compound from example 9on the scopolamine-induced cognitive deficit according to Example 56. Atthe y-axis the number of entrances into punished sector is given. At they-axis the time course in days is outlined (symbol -♦- for controlgroup, symbol -▪- for group receiving scopolamine and symbol -▴- forgroup receiving compound from exp. 9 (1 mg/kg of b.w.)+scopolamine.

FIG. 31 shows the effect of compound from example 9 on the epilepticafterdischarges elicited by stimulation of the rat somatosensory areasaccording to Example 57 at intervals 60 min and 180 min afterapplication of above mentioned compound. At the y-axis length (inseconds) and number of spike and wave afterdischarges is depicted. Atthe x-axis three pairs of columns (one column for length and second fora number of SWP) on the left side of chart stands for a dose of 10mg/kg, three pairs of columns in the right stands for a dose 0.01 mg/kgof b.w. respectively).

FIG. 32 shows the effect of compound from example 9 on the spontaneousEEG power according to Example 57 measured at intervals 60 min and 180min after application. At the y-axis the EEG power in mV² is depicted,columns at x-axis represent measurements executed before the medicationof compound from example 9 and 60 or 180 minutes after injection ofcompound from ex. 9 (1 mg/kg of b.w.).

EXAMPLES Example 1 Synthesis of 20-Oxo-5β-pregnan-3α-yl(2S)-4-(benzyloxy)-2-[(tert-butoxycarbonyl)amino]-4-oxobutanoate

The compound II (320 mg, 1 mmol) and Boc-Asp(OBzl)-OH (345 mg, 1.1 mmol)were dissolved in freshly dried benzene (35 mL). Then, about 6 mL ofbenzene was evaporated.

4-Dimethylaminopyridine (4 mg) and dicyclohexylcarbodiimide (550 mg,2.18 eq.) were added in dry benzene (3 mL) at room temperature underinert atmosphere and the reaction mixture was stirred overnight. Thereaction mixture was poured into saturated aqueous NaHCO₃ (40 mL), theproduct was extracted with EtOAc (3×30 mL) and the collected organicphases were washed 2× with water (10 mL). PrecipitatedN,N′-dicyklohexylurea was filtered off, the filtrate was dried overNa₂SO₄ and the solvent was evaporated under vacuum. Another portion ofN,N′-dicyklohexylurea was crystallised from ether, filtered off and thefiltrate containing desired product was evaporated. The residue waspurified on a column of silica gel (20 g) in a mixture of PeAe-Et₂O(9:1) to afford white foam (582 mg; 93%), [β]_(D)=+82 (c 0.36, CHCl₃).¹H NMR (400 MHz, CDCl₃): δ 0.60 (s, 3H, H-18); 0.92 (s, 3H, H-19); 1.45(s, 9H, t-Bu); 2.12 (s, 3H, H-21); 2.52 (t, 1H, J₁=8.9, H-17); 2.88 (dd,1H, J₁=16.8, J₂=4.5, H-3b′); 3.03 (bdd, 1H, J₁=16.8, J₂=4.5, H-3a′);4.50-4.55 (bm, 1H, CH-2′); 4.71-4.79 (m, 1H, H-3); 5.10-5.17 (m, 2H,H-benzyl); 5.48 (bd, 1H, J₁=8.6, HN); 7.32-7.37 (m, 5H, phenyl). ¹³C NMR(100 MHz, CDCl₃): δ 13.40 (C-18); 20.82; 22.87; 23.23; 24.38; 26.25;26.32; 26.82; 28.30 (CH₃-Boc); 31.53; 31.98; 34.57; 34.89; 35.74; 36.94;39.14; 40.38; 41.77; 44.29; 52.88; 56.62; 63.84; 66.71; 75.89; 80.02(C-Boc); 128.31 (Ar); 128.38 (Ar); 128.55 (Ar); 135.42 (Ar); 155.36(CO-Boc); 170.35, 170.70 (C-1′, C-4′); 209.60 (C-20). IR (CHCl₃): 3438(N—H, amide), 1732 (C═O, aspartate), 1702 (C═O, COCH₃, NHBoc), 1499(N—H, amide), 1232 (C—O, aspartate), 1166 (NHBoc), 1455 (ring), 1368(t-Bu). For C₃₇H₅₃NO₇ (623.8) calculated: 71.24% C, 8.56% H, 2.25% N.found: 71.40% C, 8.70% H, 2.18 N %.

Example 2 20-Oxo-5β-pregnan-3α-yl N-(tert-butoxycarbonyl)-L-aspartyl1-ester

The compound obtained in the previous Example 1 (565 mg, 0.906 mmol) wasdissolved in absolute MeOH (7 ml) and 5% Pd/CaCO₃ (56 mg) was added. Thereaction was completed after an eight-hour hydrogenation under vigorousstirring and moderate hydrogen overpressure (10 mbar). The catalyst wasfiltered off and the solvent was evaporated. Then, the product wasdissolved in ether and again evaporated to afford white foam (484 mg,100%), [α]_(D) +87.0 (c 0.49, CHCl₃). ¹H NMR (400 MHz, CD₃OD): δ 0.60(s, 3H, H-18); 0.92 (s, 3H, H-19); 1.45 (s, 9H, t-Bu-O); 2.12 (s, 3H,H-21); 2.52 (t, 1H, J₁=8.9, H-17); 2.63-2.67 (m, 2H, H-3a′,3b′); 4.45(t, 1H, H-2′); 4.65-4.73 (m, 1H, H-3). ¹³C NMR (100 MHz, CDCl₃): δ 13.40(C-18); 20.83; 22.88; 23.25; 24.40; 26.26; 26.39; 26.86; 28.30(CH₃-Boc); 31.51; 32.00; 34.60; 34.91; 35.76; 36.61; 39.13; 40.39;41.80; 44.33; 49.99; 56.63; 63.86; 76.05; 80.24 (C-Boc); 155.45 (OCONH);170.31 (C-1′); 175.22 (COOH); 209.83 (C-20). IR (CHCl₃): 3439 (N—H),1742 (C═O, aspartate, COOH, monomer), 1715 (C═O, COOH, dimer), 1703(C═O, COCH₃, NHBoc), 1500 (C—N, amide), 1369 (t-Bu-O), 1235 (C—O,aspartate), 1194 (C—O, aspartate), 1161 (NHBoc). For C₃₀H₄₇NO₇ (533.7)calculated: 67.51% C, 8.88% H, 2.62% N. found: 67.90% C, 9.01% H, 2.55%N.

Example 3 20-Oxo-5β-pregnan-3α-yl L-aspartyl 1-ester

Trifluoroacetic acid (1 mL, 13.4 mmol, 15.5 eq.) was added dropwise to astirred solution of the compound obtained in the previous Example 2 (465mg, 0.871 mmol) in dichloromethane (10 mL). The reaction mixture wasstirred for 2 h at room temperature and then it was allowed to standovernight at 5° C. Then, it was evaporated 3 times with benzene; residuewas dissolved in a mixture of pyridine (1 mL) and MeOH (1 mL), solventswere again evaporated, the residue dissolved in chloroform and washedwith water. Organic phase was dried over Na₂SO₄ and the solvent wasevaporated under vacuum. Oily residue was dissolved in ether and thesolvent was evaporated to afford white foam (370 mg, 98%), [α]_(D) +132(c 0.11, MeOH-CHCl₃ 1:1). ¹H NMR (400 MHz, CDCl₃): δ 0.60 (s, 3H, H-18);0.94 (s, 3H, H-19); 2.12 (s, 3H, H-21); 2.56 (t, 1H, J₁=8.8, H-17); 2.77(dd, 1H, J₁=17.0, J₂=8.1, H-3b′); 2.88 (bd, 1H, J₁=17.0, H-3a′);4.00-4.07 (bm, 1H, H-2′); 4.78-4.86 (bm, 1H, H-3). ¹³C NMR (100 MHz,CDCl₃-CD₃OD 1:1): δ 13.02 (C-18); 20.55; 22.53; 22.86; 24.08; 25.96;26.01; 26.58; 31.14; 31.65; 34.31; 34.55; 35.50; 35.68; 38.75; 40.12;41.58; 44.17; 50.44; 56.34; 63.57; 76.60; 169.21 (C-1′); 174.04 (COOH);210.97 (C-20). IR (KBr): 2121 (NH₃ ⁺), 1746 (C═O, aspartate), 1707 (C═O,COCH₃), 1616 (COO), 1215 (C—O, aspartate). For C₂₅H₃₉NO₅ (433.5)calculated: 69.25% C, 9.07% H, 3.23% N. found: 69.01% C, 9.23% H, 3.05%N.

The title compounds of the following Examples 4 to 12 were preparedaccording to the above-mentioned procedures of compounds that are listedat the beginning of each example.

Example 420-Oxo-5β-pregnan-3α-yl(3S)-4-(benzyloxy)-3-[(tert-butoxycarbonyl)amino]-4-oxobutanoate

Treatment of compound II (1 mmol) and Boc-Asp(OBzl) (1.1 mmol) affordedthe title compound according to the procedure described in Example 1,[α]_(D) +92.5 (c 0.24). ¹H NMR (400 MHz, CD₃OD): 0.59 (s, 3H, H-18);0.92 (s, 3H, H-19); 1.44 (s, 9H, t-Bu); 2.11 (s, 3H, H-21); 2.53 (t, 1H,J₁=8.8, H-17); 2.79 (dd, 1H, J₁=16.8; J₂=4.6, H-3b′); 2.98 (bdd, 1H,J₁=16.8; J₂=4.6, H-3a′); 4.60-4.65 (bm, 1H, CH-2′); 4.64-4.73 (m, 1H,H-3); 5.13-5.23 (m, 2H, CH₂-benzyl); 5.50 (bd, 1H, J₁=8.8 HN); 7.30-7.36(m, 5H, phenyl). IR (CHCl₃): 3439 (N—H, amide), 1717 (C═O, aspartate),1703 (C═O, COCH₃, NHBoc), 1499 (N—H, amide), 1455 (ring), 1380 (t-Bu),1163 (C—O, NHBoc). For C₃₇H₅₃NO₇ (623.8) calculated: 71.24% C, 8.56% H,2.25 N. found: 70.91% C, 8.74% H, 2.52% N.

Example 5 20-Oxo-5β-pregnan-3α-yl-N-(terc-butoxycarbonyl)-L-aspartyl4-ester

The compound from previous Example 4 afforded the title compoundfollowing the similar work-up as described in Example 2: [α]_(D) +96.3(c 0.20). ¹H NMR (400 MHz, CD₃OD): δ 0.60 (s, 3H, H-18); 0.93 (s, 3H,H-19); 1.46 (s, 9H, t-Bu); 2.11 (s, 3H, H-21); 2.52 (t, 1H, J₁=8.9,H-17); 2.81 (dd, 1H, J₁=17.0; J₂=5.2H-3b′); 3.00 (dd, 1H, J₁=16.9;J₂=4.7H-3a′); 4.56-4.64 (m, 1H, H-2′); 4.70-4.82 (m, 1H, H-3), 5.55 (d,1H, J=7.9, FIN). IR (CHCl₃): 3439 (amide), 1716 (C═O), 1703 (C═O, COCH₃,NHBoc), 1501 (amide), 1386 (t-Bu), 1232 (N—H, aspartate), 1193 (N—H,aspartate), 1160 (NHBoc). For C₃₀H₄₇NO₇ (533.7) calc.: 67.51% C, 8.88%H, 2.62% N. found: 67.25% C, 9.15% H, 2.90% N.

Example 6 20-Oxo-5β-pregnan-3α-yl L-aspartyl 4-ester

The compound from the previous Example 5 afforded the title compoundfollowing the work-up as described in Example 3, [α]_(D) +91.5 (c 0.24,MeOH-CHCl₃ 1:1). ¹H NMR (400 MHz, CDCl₃): 0.60 (s, 3H, H-18); 0.95 (s,3H, H-19); 2.12 (s, 3H, H-21); 2.54 (m, 3H, H-17); 2.86-3.08 (bm, 2H,H-3b′, H-3a′); 3.86-3.98 (bm, 1H, H-2′); 4.72-4.80 (bm, 1H, H-3); 8.00(s, 1H, HN). IR (CHCl₃): 1721 (C═O, aspartate), 1702 (C═O, COCH₃). ESIneg MS: 432.3 (100%, M−1), 415.4 (10%, M−18). For C₂₅H₃₉NO₅ (433.5)calculated: 69.25% C, 9.07% H, 3.23% N. found: 69.25% C, 9.15% H, 3.54%N.

Example 7 20-Oxo-5β-pregnan-3α-yl(2S)-5-(benzyloxy)-2-[(terc-butoxycarbonyl)amino]-5-oxopentanoate

Treatment of the compound II (1 mmol) and Boc-Glu(OBzl)-OH (1.1 mmol)afforded the title compound following the procedure descrided in Example1, [α]_(D) +70.9 (c 0.35). ¹H NMR (CDCl₃): δ 0.60 (s, 3H, CH₃-18); 0.93(s, 3H, H-19); 1.44 (s 9H, t-Bu); 2.12 (s 3H, H-21); 2.52 (t.1H, J₁=8.9,H-17); 2.88 (dd, 1H, J₁=16.8; J₂=4.5, H-3b′); 3.03 (bdd, 1H, J=16.8;J₂=4.5, H-3a′); 4.50-4.55 (bm, 1H, CH-2′); 4.71-4.79 (m, 1H, H-3);5.10-5.17 (m, 2H, H-benzyl); 5.48 (bd, 1H, J₁=8.6 HN); 7.31-7.40 (m, 5H,phenyl). ¹³C NMR (100 MHz, CDCl₃): δ 13.40 (C-18); 20.82; 22.85; 23.21;24.38; 26.23; 26.50; 26.83; 27.96; 28.29 (3×CH₃-Boc); 30.28; 31.54;32.05; 34.58; 34.89; 35.74; 39.10; 40.35; 41.78; 52.97; 56.61; 63.82;66.46; 75.67; 79.92 (C-Boc); 128.17, 128.26, 128.55 (Ph); 135.75; 155.35(OCONH); 171.65 (C-5′); 172.65 (C-1′); 209.63 (C-20). IR (CHCl₃): 3438(N—H), 1732 (C═O, glutamate), 1702 (C═O, COCH₃, NHBoc), 1499 (N—H), 1232(C—O, glutamate), 1166 (C—O, NHBoc). ESI MS: 660.5 (100%, M+Na), 638.6(14%, M), 603.5 (47%), 566.4 (39%). FIR-MS (+ESI) calcd. for C₃₈H₅₅NNaO[M+Na] 660.3871. found 660.3871. For C₃₈H₅₅NO₇ (637.8) calculated:71.55% C, 8.69% H, 2.20% N. found: 71.87% C, 8.42% H, 2.06% N.

Example 8 20-Oxo-5β-pregnan-3α-yl N-(terc-butoxycarbonyl)-L-glutamyl1-ester

The compound from the previous Example 7 afforded the title compoundfollowing the work-up as described in Example 2, [α]_(D) +87.0 (c 0.49).¹H NMR (400 MHz, CD₃OD): δ 0.60 (s, 3H, H-18); 0.92 (s, 3H, H-19); 1.45(s, 9H, t-Bu); 2.12 (s, 3H, H-21); 2.52 (t, 1H, J₁=8.9, H-17); 2.63-2.67(m, 2H, H-3a′, 3b′); 4.45 (t, 1H, H-2′); 4.65-4.73 (m, 1H, H-3). ¹³C NMR(100 MHz, CDCl₃): δ 13.40 (C-18); 20.83; 22.87; 23.22; 24.39; 26.23;26.50; 26.84; 28.09; 28.27 (3×CH₃-Boc); 30.05; 31.52; 32.05; 34.59;34.89; 35.75; 39.10; 40.37; 41.79; 52.88; 56.61; 63.83; 80.20; 75.67;80.20 (C-Boc); 155.58 (OCONH); 171.65 (C-1′); 176.59 (C-5′); 209.79(C-20). IR (CHCl₃): 3439 (N—H), 1742 (C═O, glutamate, COOH, monomer),1715 (C═O, COOH, dimer), 1703 (C═O, COCH₃, NHBoc), 1500 (C—N, amide),1369 (t-Bu), 1235 (C—O glutamate), 1194 (C—O, glutamate), 1161 (C—O,NHBoc). ESI MS: 570.5 (100%, M+Na), 648.5 (13%, M+1), 492.4 (19%), 448.4(18%). HR-MS (+ESI) calcd. for C₃₁H₄₉NNaO₇ [M+Na] 570.3401. found570.3401. For C₃₁H₄₉NO₇ (547.7) calculated: 67.98% C, 9.02% H, 2.56% N.found: 67.75% C, 9.39% H, 2.73% N.

Example 9 20-Oxo-5β-pregnan-3α-yl L-glutamyl 1-ester

The compound from previous Example 8 afforded the title compoundfollowing the similar work-up as described in Example 3: [α]_(D) +132 (c0.11, MeOH-CHCl₃ 1:1). ¹H NMR (400 MHz, CDCl₃): 0.60 (s, 3H, H-18); 0.94(s, 3H, H-19); 2.12 (s, 3H, H-21); 2.56 (t, 1H, J₁=8.8, H-17); 2.77 (dd,1H, J=17.0; J₂=8.1, H-3b′); 2.88 (bd, 1H, J₁=17.0, H-3a′); 4.00-4.07(bm, 1H, H-2′); 4.78-4.86 (bm, 2H, H-3). ¹³C NMR (100 MHz, CDCl₃-MeOD):δ 13.03 (C-18); 20.56; 22.54; 22.85; 24.08; 25.96; 26.19; 26.56; 26.97;31.14; 31.72; 33.25; 34,31; 34.54; 35.50; 38.78; 40.15; 41.56; 44.18;53.08; 56.35; 63.59; 76.43; 170.59 (C-1′); 177.67 (C-5′); 210.95 (C-20).IR (KBr): 1746 (C═O, glutamate), 1707 (C═O, COCH₃), 1215 (C—O,glutamate), 2121 (NH₃ ⁺), 1616 (CO₂ ⁻). ESI MS: 470.5 (32%, M+Na), 448.5(100%, M+1), 283.2 (49%). HR-MS (+ESI) calcd. for C₂₆H₄₁NNaO₅ [M+Na]470.2876. found 470.2877. For C₂₆H₄₁NO₅ (447.6) calculated: 69.77% C,9.23% H, 3.11% N. found: 69.46% C, 9.49% H, 3.51% N.

Example 1020-oxo-5β-pregnan-3α-yl-(4S)-5-(benzyloxy)-4-[(terc-butoxycarbonyl)amino]-5-oxopentanoate

Treatment of the compound II (1 mmol) and Boc-Glu(OBzl) (1.1 mmol)afforded the title compound following the procedure described in Example1, [α]_(D) +64 (c 0.28). ¹H NMR (CDCl₃): δ 0.60 (s, 3H, H-18); 0.93 (s,3H, H-19); 1.44 (s 9H, t-Bu); 2.12 (s, 3H, H-21); 2.38-2.5 (m, 2H,CH₂-4); 2.56 (t, 1H, J₁=8.9, H-17); 4.25-4.35 (bm, 1H, H-2′); 4.68-4.74(m, 2H, H-4); 5.10-5.17 (d, 1H, J=7.9, NH); 5.18-5.22 (m, 2H, H-benzyl);7.28-7.40 (bm, 5H, phenyl). ¹³C NMR (100 MHz, CDCl₃): δ 13.40 (C-18);20.86; 22.94; 23.26; 24.42; 26.30; 26.62; 26.90; 27.73; 28.31(3×CH₃-Boc); 30.77; 31.46; 32.18; 34.63; 34.02; 35.81; 39.21; 40.45;41.85; 52.97; 56.72; 63.88; 67.15; 74.59; 79.92 (C-Boc); 128.59, (5HPh); 135.32; 155.35 (OCONH); 172.07 (COO); 172.12 (COO); 209.43 (C-20).IR (CHCl₃): 3432 (N—H), 1730 (C═O, ester), 1702 (C═O, COCH₃, NHBoc),1496 (N—H, amide), 1451 m (ring), 1368 (t-Bu), 1164 (C—O, NHBoc). ESIMS: 660.4 (100%, M+Na). HR-MS (+ESI) calcd. for C₃₈H₅₅NNaO [M+Na]660.3871, found 660.3870. For C₃₈H₅₅NO₇ (637.8) calculated: 71.55% C,8.69% H, 2.20% N. found: 71.27% C, 8.75% H, 2.32% N.

Example 11 20-Oxo-5β-pregnan-3α-yl N-(terc-butoxycarbonyl)-L-glutamyl5-ester

Compound from Example 10 afforded the title compound following theprocedure of Example 2, [α]_(D) +80.0 (c 0.31). ¹H NMR (400 MHz, CD₃OD):δ 0.63 (s, 3H, H-18); 0.99 (s, 3H, H-19); 1.45 (s, 9H, t-Bu); 2.13 (s,3H, H-21); 2.52 (t, 1H, J₁=8.9, H-17); 2.87 (dd, 1H, J=10.2, J′=3,1H-3b′); 3.04 (dd, 1H, J=10.2, J′=3, 1H-3a′); 4.47 (t, 1H, H-2′);5.22-5.28 (m, 1H, NH); ¹³C NMR (100 MHz, CDCl₃): δ 13.32 (C-18); 20.80;22.86; 23.18; 24.34; 26.22; 26.54; 26.84; 27.65; 28.23 (3×CH₃-Boc);30.84; 31.40; 32.10; 34.60; 34.96; 35.75; 39.12; 40.38; 41.80; 44.32;52.84; 56.65; 63.86; 74.66; 80.81; 80.20 (C-Boc); 155.67 (OCONH); 171.58(C-1′); 174.14 (C-5′); 210.13 (C-20). IR (CHCl₃): 3436 (N—H, amide),1730 (C═O, ester), 1710 (C═O, NEBoc), 1700 (C═O, COCH₃), 1501 (N—H),1163 (C—O, NHBoc). ESI neg. MS: 546.5 (100%, M−1), 472.4 (10%). HR-MS(+ESI) calcd. for C₃₁H₄₈NO₇ [M−H] 546.3436, found 546.3436. ForC₃₁H₄₉NO₇ (547.7) calculated: 67.98% C, 9.02% H, 2.56% N. found: 67.59%C, 9.23% H, 2.93% N.

Example 12 20-Oxo-5β-pregnan-3α-yl-L-glutamyl 5-ester

The compound from previous Example 11 afforded the title compoundfollowing the procedure described in Example 3, [α]_(D) +93.7 (c 0.26,MeOH-CH₃OH 1:1). ¹H NMR (400 MHz, CDCl₃): 0.60 (s, 3H, H-18); 0.94 (s,3H, H-19); 2.12 (s, 3H, H-21); 2.56 (t, 1H, J₁=8.8, H-17); 2.50-2.60 (m,1H, H-3a′); 3.85-3.87 (m, 1H, H-2′); 4.78-4.86 (bm, 1H, H-3); 7.35-7.40(m, 1H, NH). ¹³C NMR (100 MHz, MeOD): δ 13.08 (C-18); 22.02; 23.79;23.94; 25.48; 27.49; 27.57; 27.67; 28.14; 31.56; 31.66; 33.40; 35.81;36.16; 37.26; 40.28; 41.83; 43.37; 45.48; 55.50; 57.93; 64.90; 76.20;174.06 (C-11; 176.13 (C-5′); 212.47 (C-20). IR (CHCl₃): 1732 (C═O,glutamate), 1703 (C═O, COCH₃), 1215 (C—O, glutamate), 1616 (COO). ESIMS: 446.4 (100%, M−1). HR-MS (+ESI) calcd. for C₂₆H₄₀NO₅ [M−1] calcd.446.2912, found 446.2912. For C₂₆H₄₁NO₅ (447.6) calculated: 69.77% C,9.23% H, 3.13% N. found: 69.39% C, 9.53% H, 3.40% N.

Example 13 20-Oxo-5β-pregnan-3α-yl-adipyl 1-ester

Dicyclohexylcarbodiimide (410 mg, 2 mmol) in dry benzene (10 mL) wasadded into a solution of adipic acid (300 mg, 2 mmol) in dry THF (10 mL)under inert atmosphere and the mixture was stirred for 1 hour. Then asolution of compound H (3α-hydroxy-5β-pregnan-20-one) (320 mg, 1 mmol)and dimethylaminopyridine (10 mg, 0.08 mmol) in dry benzene (10 mL) wasadded dropwise over 15 min. This reaction mixture was stirred at roomtemperature for 16 hours and then, the solvents were evaporated. Theresidue was purified on a column of silica gel (PeAe/Et₂O, 9:1) toafford non-crystallizable hemiester (350 mg, 78%), [α]_(D) +89 (c 0.49).¹H-NMR: δ 0.60 (s, 3H, (H-18); 0.92 (s, 3H, H-19); 2.11 (s, 3H, H-21);2.30 (m, 2H, W˜20, H-adipate); 2.37 (m, 2H, W˜20, H-adipate); 2.53 (t,1H, J=9, H-17); 4.73 (m, 1H, W=35, H-3). IR (CHCl₃): 1727, 1706 (C═O);1358 (CH₃C═O); 1233, 1193, 1183 (C—O). For C₂₇H₄₂O₅ (446.6) calculated:72.61% C, 9.48% H. found: 72.13% C, 9.53% H.

Compounds described in Examples 14-16 were prepared according the sameprocedure as in Example 13 with the fact that instead of adipic acid apimelic acid (heptanedioic acid) was used in Example 14; suberic acid(cork, octandioic acid) was used in Example 15 and fumaric acid (2(E)-butene-dioic acid) was used in Example 16.

Example 14 20-Oxo-5β-pregnan-3α-yl 6-carboxyhexanoate

Oily, [α]_(D) +72.3 (c 0.33). ¹H-NMR: δ 0.60 (s, 3H, (H-18); 0.93 (s,3H, H-19); 2.11 (s, 3H, H-21); 2.30 (m, 2H, W˜20, H-pimelate); 2.37 (m,2H, W˜20, H-pimelate); 2.53 (t, 1H, J=8.8); 4.74 (m, 1H, W=35, H-3). ¹³CNMR (100 MHz, MeOD): δ 13.40 (C-18); 20.86; 22.92; 23.28; 24.42; 24.70;24.74; 26.33; 26.69; 26.92; 28.66; 31.48; 32.28; 34. 55; 34.64; 35.07;35.82; 39.22; 40.44; 41.86; 44.33; 44.84; 56.72; 63.90; 74.06; 173.19(COO—); 178.59 (COO); 209.59 (C-20). IR (CHCl₃): 3516 (COOH, monomer),1725 (C═O, ester), 1705 (C═O, COCH₃), 1261 (C—O, ester). MS: (ESI): 460(4%, M). HR-MS (+ESI) calcd. for C₂₈H₄₄O₅Na [M+Na] 483.3081, found483.3080.

Example 15 20-Oxo-5β-pregnan-3α-yl 7-carboxyheptanoate

Oily, ¹H-NMR: δ 0.60 (s, 3H, (H-18); 0.92 (s, 3H, H-19); 2.11 (s, 3H,H-21); 2.30 (m, 2H, W˜20, H-suberate); 2.37 (m, 2H, W˜20, H-suberate);2.54 (t, 1H, J=8.7); 4.73 (m, 1H, W=35, H-3). ¹³C NMR (100 MHz, MeOD): δ13.40 (C-18); 20.86; 22.93; 23.28; 24.42; 24.50; 24.83; 26.33; 26.70;26.92; 28.67; 28.70; 31.48; 32.30; 33.77; 34. 63; 34.65; 35.06; 35.82;39.22; 40.45; 41.86; 44.33; 56.74; 63.90; 74.06; 173.19 (COO—); 178.59(COO); 209.60 (C-20). IR spectrum (CHCl₃): 3517 (COOH, monomer), 1725(C═O, ester), 1708 (C═O, COCH₃), 1194 (C—O, ester), 1616 (COO). ESI MS:497.4 (100%, M+Na). HR-MS (+ESI) calcd. for C₂₉H₄₆NaO₅ [M+Na] 497.3237,found 497.3239.

Example 16 20-Oxo-5β-pregnan-3α-yl (E)-3-carboxyprop-2-enoate

¹H-NMR: δ 0.62 (s, 3H, H-18); 0.99 (s, 3H, H-19); 2.11 (s, 3H, H-21);2.53 (t, 1H, J=8, H-17); 4.77 (m, 1H, W=35, H-3); 6.69 (d, 2H, J=14,H-fumarate). IR (CHCl₃): 1723, 1702 (C═O); 1358 (CH₃C═O); 1181, 982(C—O); 1645 (C═C). For C₂₅H₃₆O₅ (416.6) calculated: 72.08% C, 8.71% H.found: 72.05% C, 8.86% H.

Example 17 20-Oxo-5β-pregnan-3α-yl (Z)-3-carboxyprop-2-enoate

4-Dimethylaminopyridine (1 mg) a maleic anhydride (180 mg, 1.55 mmol)were added into a solution of the compound II (100 mg, 0.31 mmol) inpyridine (0.5 mL) at 0° C. The reaction mixture was allowed to stand at40° C. for 16 h and then poured into ice. The product was extracted withEtOAc (3×30 mL), collected organic phases were washed with aqueoussolution of citric acid (1%, 30 mL), aqueous solution of sodiumbicarbonate (5%, 20 mL), and water (30 mL). Organic phase was dried oversodium sulfate and solvents were evaporated. The residue was purified onprep-TLC (200×200×0.3 mm) to afford 116 mg (90%) of hemiester, ¹H-NMR: δ0.62 (s, 3H, H-18); 0.99 (s, 3H, H-19); 2.11 (s, 3H, H-21); 2.67 (t, 1H,J=9, H-17); 4.77 (m, 1H, W=35, H-3); 6.77 (d, 2H, J=6, H-maleinate). ¹³CNMR (100 MHz, CD₃OD): δ 13.80 (C-18); 22.03; 23.80; 23.93; 25.48; 27.47;27.54; 28.14; 33.14; 35.83; 36.15; 37.26; 40.25; 41.80; 43.39; 45.47;57.86; 64.89; 76.81; 130.66; 131.66; 166.96; 212.45. IR (CHCl₃): 1725,1705 (C═O); 1358 (CH₃C═O); 1172, 982 (C—O); 1645 (C═C). HR-MS (ESI)calculated for C₂₅H₃₆O₅ (M+Na) 439.2455 found 439.2455. For C₂₅H₃₆O₅(416.6) calculated: 72.08% C, 8.71% H. found: 72.05% C, 8.86% H.

Compounds in Example 18 and 19 were preparded according to the procedureof Example 17.

Example 18 20-Oxo-5β-pregnan-3α-yl 3-carboxyperfluoropropanoate

Oily material was obtained by a treatment of compound II (240 mg, 0.75mmol) in pyridine (5 mL) and perfluorosuccinanhydride (645 mg, 3.75mmol). ¹H-NMR: δ 0.60 (s, 3H, H-18); 0.92 (s, 3H, H-19); 2.11 (s, 3H,H-21); 2.53 (t, 1H, J=8.7); 4.75 (m, 1H, W=35, H-3). This compound wascharacterized as methylester: [α]_(D)=+82.8 (c 0.21, MeOH). ¹H-NMR: δ0.61 (s, 3H, H-18); 0.95 (s, 3H, H-19); 2.12 (s, 3H, H-21); 2.55 (t, 1H,J=8.9, H-17); 3.10 (s, 3H, methylester); 4.98 (m, 1H, W=35, H-3); 1775,1699 (C═O); 1357 (CH₃C═O); 1180, 1154, 1140, 1103 (C—O, C—F₂). ESI MS:527.0 (100%, M+Na). HR-MS (ESI) calculated for C₂₆H₃₆F₄O₅Na (M+Na)527.2391 found 527.2388.

Example 19 20-Oxo-5β-pregnan-3α-yl 3-carboxy-ξ-methyl-propanoate

A title compound (a mixture of isomers) was obtained by a treatment ofcompound II (100 mg, 0.31 mmol), 4-dimethylaminopyridine (3 mg, 0.03mmol), and methyl-succinyl-anhydride (170 mg, 1.5 mmol). ¹H-NMR: δ 0.60(s, 3H, H-18); 0.93 (s, 3H, H-19); 2.11 (s, 3H, H-21); 2.30 (m, 2H,H-methylpropanoate); 2.55 (t, 1H, J=8, H-17); 4.75 (m, 1H, w=35, H-3).IR (CHCl₃): 1712, 1702 (C═O); 1358 (CH₃C═O); 1181, 987 (C—O). ForC₂₆H₄₀O₅ (432.6) calculated: 72.19% C, 9.32% H. found: 71.88% C, 9.70%H.

Example 20 Compound HET a)4-[1,11-Bis(methylensulfo)-3,3,9,9-tetramethyl-4,8-diaza-6-oxa-3,6,8,9-tetrahydropentacen-13-yl]-benzene-1,3-dicarboxylicacid 1-succinimidyl ester

Active ester Alexa fluor 567 (Molecular Probes) (57.3 mg, 64.1 μmol) andaspartate from Example 3 (33 mg, 1.2 eq., 76.8 μmol) were dissolved inDMSO (3 mL) and pyridine (1 mL) and the reaction mixture was treated at40° C. for 72 h. The completion of the reaction was checked by HPLC andsolvents were evaporated under vacuum at 60° C. The residue was purifiedon preparative TLC in a mixture of butanol-methanol-water (3:1:1) and 1%(v/v) of triethylamine. The eluate was dried under reduced pressure (1mm) to afford 40.3 mg of the title compound IR: 1648 s (CONH), 3360,3300 (NH), 1246 (C—O, ester), 1731 (C═O, ester), 1695 (C═O, ketone),1718, 1685 (C═O, acid), 1229 (SO₃H), 1612, 1497, 1405 (ring). MS-ESIneg: 368.8 (63%, M−3H)⁻³, 553.7 (63%, M−2H)⁻², 1130.1 (2%, M+Na), 1146.1(6%, M+K); (HR) for (C₅₈H₆₇N₃O₁₅N₂-2H)⁻² calculated: 553.69286. found:553.69231. ¹H NMR (400 MHz, D₂O, K₂CO₃): 0.54 (s, 3H, H-18); 0.90 (s,3H, H-19); 0.88-0.92 (m, 4CH₃, rhodamine) 1.27 (t, 9H, J=7.0, NCH₂CH ₃);2.12 (s, 31-1, H-21); 3.60 (t, 8H, J=7.0, NCH ₂CH₃); 5.69 (s, 2H,H-11+21, rhodamine); 6.53 (m, 4H, 4H-rhodamine); 7.33 (d, 2H, J=8.0H-6′and H-2′, rhodamine); 8.19 (d, 2H, J=8.0, H-3′ a H-5′, rhodamine).

b) 20-Oxo-5β-pregnane-3α-ylN-(7-nitrobenz-2-oxa-1,3-diazole-4-yl)-L-aspartyl 1-ester

Steroid conjugate from example 3 (103 mg; 0.24 mmol) was dissolved indry dichloromethane (7 mL) and triethylamine (0.10 mL, 0.72 mmol) andsolution of NBD-Cl (57 mg, 0.29 mmol) in dichloromethane (1 mL) wasadded at room temperature. The mixture was stirred overnight in darknessand then poured into water (20 mL), acidified by aq. HCl to pH 3-4 andorganic phase was separated. Aqueous phase was then extracted with EtOAc(3×10 mL), washed with brine and dried with anhydrous MgSO₄. Solventswere evaporated in vacuo and the residue was purified by preparative TLCin MeCN:MeOH:AcOH (90:10:1) to afford title compound as orange-brownfoam (121 mg, 85%). [α]_(D)=+39 (c 0.05, CHCl₃). NMR (400 MHz, CDCl₃): δ0.60 (s, 3H, H-18); 0.94 (s, 3H, H-19); 2.12 (s, 3H, H-21); 2.54 (t.1H,J₁=8.7, H-17); 3.14 (dd, 2H, J₁=17.1, J₂=5.0, H-3′); 4.79-4.83 (m, 1H,2′); 4.83-4.91 (m, 1H, H-3); 6.29 (d, 1H, J=8.5; H—N); 7.01 (d, 1H,J₁=8.4, Ar); 8.50 (d, 1H, J=8.5 Ar). IR (CHCl₃): 1736 (C═O, ester), 1699(C═O, COCH₃), 1499 (N—H, amide), 1572, 1318 (NO₂). ESI MS: 641.3 (22%,M+2Na+H), 619.3 (100%, M+Na), 597.1 (9%, M+H).

Example 21 20,20-(Ethylendioxy)-5β-pregnan-3β-yl-tosylate

A solution of 20-oxo-5β-pregnan-3β-yl-tosylate (Swann D. A., Turnbull J.H.: Tetrahedron 1966, 22, 231; 200 mg, 0.42 mmol), orthoethyl formate(0.38 ml, 2.3 mmol), ethylene glykol (0.3 ml, 5.8 mmol), andp-toluensulfonic acid monohydrate (2 mg, 0.01 mmol) in dry benzene (2mL) were stirred at room temperature for 2 days. The reaction mixturewas poured into saturated solution of sodium bicarbonate (30 mL),steroid was extracted with EtOAc (2×60 mL), the organic phase was washedwith water and dried over magnesium sulfated. Solvents were evaporatedand the residue was purified on prep-TLC (6 plates, 200×200×0.3 mm) in amixture of PeAe/Et₂O (1:1) and 2 drops of pyridine to afford 194 mg(89%) of title compound, m.p. 146-148° C. (PeAe-Et₂O), [α]_(D) +11.7 (c0.37, CHCl₃). ¹H-NMR (200 MHz): 0.73 (s, 3H, H-18); 0.94 (s, 3H, H-19);1.27 (s, 3H, H-21); 2.44 (s, 3H, CH₃, tosylate); 3.81-4.01 (m, 4H,OCH₂CH₂O); 4.83 (m, 1H, H-3); 7.32 (d, 2H, J=7.8, H-3 and H-5,tosylate); 7.78 (d, 2H, J=8.3, H-2 and H-6, tosylate). IR (CHCl₃): 1358(SO₂, tosylate), 1189 (SO₂, tosylate), 1053 (OCH₂CH₂O—), 901 (C—O,tosylate). FAB MS: 517 (6%, M+H), 345 (2.5%, M-tosylate). For C₃₀H₄₄O₅S(516.7) calculated: 69.73% C, 8.58% H, 6.21% S. found: 69.92% C, 8.79%H, 6.41% S.

Example 22Dimethyl-(2-[20,20-(ethylendioxy)-5β-pregnan-3α-yl]propandioate)

To a stirred solution of sodium salt of dimethyl malonate, prepared byrefluxing toluene (30 mL), sodium (80 mg, 3.5 mmol), and dimethylmalonate (0.707 mL, 6.2 mmol) until all the sodium had been dissolved,was added a solution of compound from Example 21 (600 mg, 1.16 mmol) intoluene (20 mL) dropwise during vigorous stirring. After refluxing foradditional 12 hours, the solution was cooled to room temperature, theprecipitated sodium p-toluenesulfonate was filtered off, washed withsmall amount of toluene, and the combined toluene extracts wereevaporated. The obtained oily residue was dissolved in ether (200 mL)and washed with water, and the solution was dried, and evaporated. Theresidue was purified by chromatography on a column of silica gel (20 g)in a mixture of PeAe/Et₂O (9:1) to afforded title compound (430 mg,77%): m.p. 98-102° C. (ethyl acetate), [α]_(D) +55.4 (c 0.25, CHCl₃). ¹HNMR (400 MHz, CDCl₃): 0.73 (s, 3H, H-18); 0.93 (s, 3H, H-19); 1.29 (s,3H, H-21); 3.19 (d, 1H, J=9.2, CH(COOMe)₂); 3.72 (s, 6H, COOMe);3.80-4.02 (m, 4H, —OCH₂CH₂O—). IR (CHCl₃): 1753 (C═O, COOMe), 1731 (C═O,COOMe); 1436 (CH₃, COOMe); 1472, 1295 (CH₂, acetal); 1149 (C—O). ESI MS:976 (47%, 2M+H+Na), 975 (100%, 2M+Na), 499 (15%, M+Na). For C₂₈H₄₄ ^(O)₆ (476.6) calculated: 70.56% C, 9.30% H. found: 70.61% C, 9.53% H.

Example 23 Methyl-(2-[20,20-(ethylendioxy)-5β-pregnan-3α-yl]-acetate)

A solution of compound from Example 22 (187 mg, 0.39 mmol) and sodiumcyanide (37.5 mg, 0.76 mmol) in dimethylsulfoxide (11 mL) were heatedunder inert atmosphere at 210° C. for 3 hours. After cooling, themixture was poured into water (100 mL) and extracted with ether (3×100mL). The collected organic phases were washed with water and taken down.The residue was purified by prep-PLC (6 plates) in a mixture ofPeAe/Et₂O (4:1), affording 90 mg (57%) of title compound, m.p. 75-76° C.(ethyl-acetate). [α]_(D) +68.0 (c 0.25, CHCl₃). ¹H-NMR (400 MHz, CDCl₃):0.73 (s, 3H, H-18); 0.93 (s, 3H, H-19); 1.29 (s, 3H, H-21); 2.01 (m, 1H,H-3β); 2.22 (d, 2H, J=7.3, CH ₂COOCH₃); 3.66 (s, 3H, COOCH ₃); 3.85-4.00(m, 4H, —OCH₂CH₂O—). IR (CHCl₃): 1729 (C═O), 1295, 1073, 1054 (CH₂,acetal), 1142 (C—O). ESI MS: 419 (61%, M+1); 357 (26%, M-CH₂COOCH₃); 230(100%). For C₂₆H₄₂O₄ (418.6) calculated: 74.60% C, 10.11% H. found:74.42% C, 10.30% H.

Example 24 2-[20,20-(Ethylendioxy)-5β-pregnan-3α-yl]acetic acid

A solution of potassium hydroxide (55 mg, 0.98 mmol) in ethanol (0.125mL) and water (0.125 mL) was added to a stirred solution of compoundfrom Example 23 (75 mg, 0.18 mmol) in methanol (2 mL). The mixture washeated at 120° C. for 1.5 hour. After cooling, it was poured into water(50 mL), an aqueous solution of hydrochloric acid (12%) was added to theaqueous phase to reach pH 1, and the product was extracted with ether(30 mL) and chloroform (2×30 mL). Collected organic phases were washedwith water, dried, and evaporated to give title compound (45 mg, 63%),m.p. 164-167° C., [α]_(D) +41.0 (c 0.19, CHCl₃). ¹H NMR (400 MHz,CDCl₃): 0.74 (s, 3H, H-18); 0.93 (s, 3H, H-19); 1.29 (s, 3H, H-21); 2.02(m, 1H, H-3β); 2.26 (d, 2H, J=6.6, CH ₂COOH); 3.86-3.98 (m, 4H,—OCH₂CH₂O—). IR (CHCl₃): 3516 (O—H, COOH, monomer), 3088 (O—H, COOH,dimer), 1739 (C═O, COOH, monomer), 1705 (C═O, COOH, dimer), 1496, 1375,1054 (CH₂, acetal). FAB MS: 405 (4%, M+H); 261 (1%, M-CH₂COOH, C₄H₇O₂);231 (2%, M-CH₂COOH, C₄H₇O₂, 2×CH₃). For C₂₅H₄₀O₄ (404.2) calculated:74.22% C, 9.97% H. found: 73.99% C, 10.16% H.

Example 25 a) 2-(Oxo-5β-pregnan-3α-yl)-acetic acid

A solution of p-toluensulfonic acid monohydrate (20 mg, 0.12 mmol) inwater (0.6 mL) was added into a solution of compound from Example 24 (55mg, 0.13 mmol) in acetone (5 mL) and the reaction mixture was stirredfor 24 hours. Then, it was poured into water, acidified by an aqueoussolution of hydrochloric acid (12%), and extracted with ether (30 mL)and chloroform (2×30 mL). Collected organic phases were washed withwater, dried, and evaporated to give title compound (42 mg), m.p.189-192° C. (acetone), [α]_(D) +127.6 (c 0.2, CHCl₃). IR (CHCl₃): 3516(O—H, COOH, monomer), 3090 (O—H, COOH, dimer), 1739 (C═O, COOH,monomer); 1702 (C═O, COOH, dimer a C═O, ketone), 1702 (C═O, COCH₃). ¹HNMR (400 MHz, CDCl₃): 0.59 (s, 3H, H-18); 0.93 (s, 3H, H-19); 2.11 (s,3H, H-21); 2.16 (m, 1H, H-3β); 2.27 (d, 2H, J=6.8, CH ₂COOH); 2.54 (t,1H, J=8.8, H-17). ESI MS: 720 (42%, 2M), 719 (100%, 2M−1), 359 (21%,M−1). For C₂₃H₃₆O₃ (360.3) calculated: 76.62% C, 10.06% H. found: 76.54%C, 10.25% H.

b) 2-(20-Oxo-5β-pregnan-3α-yl)propandioic acid

A solution of potassium hydroxide (140 mg, 2.5 mmol) in ethanol (0.35mL) and water (0.35 mL) was added to a stirred solution of dimethylesterfrom Example 22 (55 mg, 0.11 mmol) in methanol (4 mL). The mixture washeated at 120° C. for 6 hours. After cooling, it was poured into waterand extracted with ether (50 mL). A mixture of HCl/H₂O (1:2) was addedto the aqueous phase up to pH 1 and it was allowed to stay at roomtemperature for 3 hours. Subsequently, it was extracted with chloroform(2×30 mL), collected organic phases were washed with water, dried andevaporated. The residue was crystallized from PeAe/Et to give titlecompound (27 mg, 58%): m.p. 214-215° C., [α]_(D) +82.7 (c 0.25, CHCl₃).¹H NMR (400 MHz, CDCl₃): 0.58 (s, 3H, H-18); 0.93 (s, 3H, H-19); 2.12(s, 3H, H-21); 2.15 (m, 1H, H-3β); 2.54 (t, 1H, J=8.6, H-17); 3.17 (d,1H, J=8.5, CH(COOH)₂). IR (CHCl₃): 3500 (O—H, COOH, monomer), 3200 (O—H,COOH, dimer), 1741 (C═O, COOH, monomer), 1702 (C═O, ketone). ESI MS: 808(4%, 2M), 404 (22%, M), 403 (100%, M−H). For C₂₄H₃₆O₅ (404.5)calculated: 71.26% C, 8.97% H. found: 71.08% C, 8.80% H.

Example 26 N-(20-Oxo-5β-pregnan-3α-yl) glycine methylester hydrochloride

Glycine methyl ester hydrochloride (89 mg, 0708 mmol) with triethylamine(0.072 mL) and 5β-pregnane-3,20-dione (200 mg, 0.632 mmol) were mixed inneat titanium (IV) isopropoxide (302 mg, 1.06 mmol) and dry toluene (0.4mL). The mixture was stirred under nitrogen for 3 h. Methanol (2.8 mL)was added. Then NaBH₄ (38 mg, 1.01 mmol) was carefully added. After 5min the reaction was finished by adding 0.1 N NaOH. The resultingmixture was filtered through Celite, and the residue was washed withether (2×30 mL) and ethyl acetate (2×30 mL). The organic layer wasseparated, dried (MgSO₄) and the solvents were removed under reducedpressure. The residue was purified on preparative TLC (5 plates,200×200×0.3 mm) in a mixture of toluene-ethyl acetate (1:1) to afford 43mg (17%). ¹H NMR (400 MHz, CDCl₃): δ 0.59 (s, 3H, H-18); 0.92 (s, 3H,H-19); 2.11 (s, 3H, H-21); 2.45-2.53 (m, 1H, H-3); 2.52 (t, 1H, J=8.8,H-17); 3.46 (s, 2H, H-2′); 3.74 (s, 3H, OMe). ¹³C NMR (100 MHz, CDCl₃):δ 13.34 (C-18); 20.77; 22.86; 23.45; 24.40; 26.37; 27.15; 27.87; 31.45;34.00; 35.08; 35.69; 35.83; 39.22; 40.29; 42.09; 44.27; 48.09; 51.74;56.70; 57.24; 63.86; 173.13 (C-1′); 209.51 (C-20). The product wassubsequently characterised as hydrochloride. [α]_(D)=+61.9 (c 0.24,CHCl₃). IR (CHCl₃): 2708 (N⁺H₂), 1758 (C═O, ester), 1698 (C═O, COCH₃).1235 (C-0, ester). MS-ESI: 390.2 [100%, M+H]⁺; (HR) for [C₂₄H₄₀NO₃+H]⁺m/z calculated: 390.3003. found: 390.3002.

Example 27 N-(20-Oxo-5β-pregnan-3α-yl) glycine

Compound from the example 26 (73 mg; 187 μmol) was dissolved in dryethylacetate (1.9 mL) along with lithium iodide (150 mg; 1.12 mmol) andthe mixture was refluxed 2 days under argon atmosphere. The reactionmixture was then finished by addition of water (2 mL), product wasextracted with chloroform (3×2 mL). Organic layer was washed with brine(2 mL), dried over MgSO₄ and evaporated to give crude title acid, whichwas purified on preparative TLC (2 plates) in a mixture ofchloroform-methanol-triethylamine (180:20:1) to afford the off-whitesolid (20 mg; 29%). ¹H NMR (400 MHz, CDCl₃-MeOD 4-1): δ 0.61 (s, 3H,H-18); 0.99 (s, 3H, H-19); 2.14 (s, 3H, H-21); 2.60 (t.1H, J=8.8, H-17);3.08-3.16 (m, 1H, H-3); 3.49 (s, 2H, H-2′). IR (CHCl₃): 3452 (NH) 2650,2470 (N⁺H₂), 1698 (CH₃C═O, —COOH), 1637 (N⁺H₂), 1472 (N⁺H₂), 1390 (CH₃),1358 (CH ₃CO). MS-ESI: 376.2 [100%, M+H]⁺; 398.2 [7%, M+Na]⁺ (HR) for[C₂₃H₃₇O₃N+H]⁺ m/z calculated: 376.2846. found: 376.2846.

Example 28 Effects of Pregnanolone Sulphate and its Analogues on Nativeand Recombinant NMDA Receptors

Primarily dissociated hippocampal cultures were prepared from1-2-day-old rat pups. Animals were decapitated and subsequentlyhippocampus was isolated. Cell suspension was prepared by trypsinetreatments and mechanical dissociation. The cells were inoculated on 12-and 25-mm polylysine-coated glasses at a density of approx. 500 000cells/cm². Neuronal cultures were maintained in Neurobasal™-A medium(Invitrogen, Carlsbad, USA) with glutamine (0.5 mM) a B-27 Serum-FreeSupplement (Invitrogen) at 37° C. and 5% CO₂.

HEK293 cells (American Type Culture Collection, ATTC No. CRL1573,Rockville, Md.) were cultivated in Opti-MEM® I media (Invitrogen) withaddition of 5% fetal bovine serum at 37° C. and transfected withNR1-1a/NR2B/GFP plasmids, as described in the scientific literature(Cais et al., 2008). Same amounts (0.3 μg) of cDNA coding NR1, NR2 andGFP (green fluorescent protein) (pQBI 25, Takara, Japan) were mixed with0.9 μl of Matra-A Reagent (IBA, Gottingen, Germany) and added toconfluent HEK293 cells cultivated in v 24-pit cultivating plate. P Aftertrypsination, the cells were re-suspended in Opti-MEM® I containing 1%fetal bovine serum. Subsequently, 20 mM MgCl₂, 1 mMD,L-2-amino-5-phosphonopentanoic acid, 3 mM kynurenic acid and ketaminewas added to the mixture and cells were inoculated on thepolylysine-coated glass plates having 25 mm in diameter. The followinggenes coding NMDA receptor subunits were used for transfection: NR1-1a(GenBank accession no. U08261) and NR2B (GenBank accession no. M91562).

5-10-day old hippocampal culture cells or HEK293 cultured cells wereused for electrophysiological investigations with a latency of 16-40 hafter transfection. Whole-cell currents were measured by patch-clampamplifier (Axopatch 1D; Axon Instruments, Inc. Foster City, USA) aftercapacitance and serial resistance (<10 MΩ) compensation to 80-90%.Agonist-induced responses were filtered to 1 kHz (8-pole Bessel filter;Frequency Devices, Haverhill, USA), digitized with sampling frequency of5 kHz and analyzed by pClamp v.9 software (Axon Instruments, USA).Micropipettes made of borosilicate glass were filled with intracellularsolution, containing 125 mM D-glukonic acid, 15 mM cesium chloride, 5 mMEGTA, 10 mM HEPES buffer, 3 mM magnesium chloride, 0.5 mM calciumchloride and 2 mM magnesium-salt of ATP (pH adjusted to 7.2 by cesiumhydroxide solution). Extracellular solution (ECS) contained 160 mMsodium chloride, 2.5 mM potassium chloride, 10 mM HEPES, 10 mM glucose,0.2 mM EDTA a 0.7 mM calcium chloride (pH adjusted 7.3 by sodiumhydroxide solution). Glycine was added to both testing and controlsolution. Moreover, bicuculline (10 μM) and tetrodotoxin (0.5 μM) wasadded to hippocampal cultures. Steroid-containing solutions wereprepared from fresh solution (20 mM) of steroid dissolved indimethyl-sulfoxide (DMSO). Same concentrations of DMSO were used in allextracellular solutions. Control and experimental solutions were appliedvia microprocessor-controlled perfusion system with approx. rate ofsolution exchange in areas adjacent to cells reaching ˜10 ms.

Current responses produced by 100 μM NMDA (for hippocampal neurons) or 1mM glutamate (for recombinant NMDA receptors) were measured at membranepotential maintained at −60 mV. Similarly as described before,pregnanolon sulphate decreased the amplitude of responses elicited byNMDA. After application of 100 μM preganolone sulphate the meaninhibition effect reached 71.3±5.0% (n=5) of responses elicited by NMDAreceptor activation on hippocampal neurons and 67.2±8.2% (n=5) onrecombinant NR1/NR2B receptors (Petrovic et al., 2005). Our syntheticanalogues of pregnanolone sulphate exhibited significant inhibitoryeffect (30-70% of maximum inhibition) at concentration range 50, 100 and200 μM. Relative effect of steroid-induced inhibition was used forcalculating IC₅₀. IC₅₀ was calculated using formulaRI=1−(1/1+([steroic]/IC₅₀)^(h)), where RI denotes relative effect ofsteroid-induced inhibition and h is a parameter of Hill's coefficient(1.2). IC₅₀ values are stated in the following table.

Newly synthesized analogues from Examples 1-25 have the same mechanismof action on the NMDA receptors as pregnanolone sulphate, but theydiffer in their relative affinities (see Table 1).

TABLE 1 Tested compound - Relative Compound inhibition IC₅₀ Number fromexample No. effect (%) (μmol) of cells Pregnanolone sulphate 67.2 ± 8.2 55 5 Compound from Example 26 43.4 ± 3.4  250 4 Compound from Example 2563.6 ± 21.1 126 5 Compound from Example 3  83.6 ± 2.7  51 5 Compoundfrom Example 9  77.1 ± 5.2  73 5 Compound from Example 20 36.3 ± 3.2 320 5 Compound from Example 13 87.3 ± 8.9  40 4 Compound from Example 1781.6 ± 2.7  14 6 Compound from Example 19 80.9 ± 3.6  30 4 Compound fromExample 15    54.0 ± 10.8% 175 5 Compound from Example 14    61.5 ±10.6% 135 5

The results show that synthetic analogues of pregnanolone sulphate havethe same mechanism of action on NMDA receptors as pregnanolone sulphate;however, they differ in their affinity for these receptors. Due toethically-justified requirements for minimization of numbers oflaboratory animals used in experimental studies, we have chosen thecompound from the Example 9 as a model molecule and we have subjected itto intimate investigation regarding its effect on animal behavior andits neuroprotective properties in animal models. All experimentscomplied with standard accepted rules for animal care (Animal ProtectionCode of the Czech Republic, EU directives, and National Institute ofHealth guidelines).

Example 29

Effect of the compound from Example 9 on spontaneous locomotor activityin the open-field test and sensorimotor gating tested with prepulseinhibition of the startle response

For assessment of the spontaneous locomotor activity, animals wereobserved for 30 min in a square open-field apparatus (1 m×1 m). Animalswere tracked with Ethovision Pro system (Noldus, Netherlands) and totalpath traveled during this session was evaluated. The compound fromExample 9 was applied subcutaneously (dissolved in cyclodextrin) 30 minprior to behavioral observations at doses 1 mg/kg a 10 mg/kg; controlanimals were injected with saline.

Results showed that compound from Example 9 did not significantly alterthe spontaneous locomotor activity; all groups exhibited similar totaldistance (FIG. 2; top panel).

Testing prepulse inhibition of the startle response as a measure ofpossible deficit in sensorimotor gating has shown that none of the dosesof the compound from Example 9 significantly altered this parameter(compared to control animals (FIG. 2; bottom panel).

Example 30

Effect of compound from Example 9 and action of dizocilpine (anon-competitive antagonist of NMDA receptors; standardly used aspositive control and referred hereafter as dizocilpine) on the behaviorof rats in active allothetic place avoidance (AAPA), a spatial taskrequiring spatial orientation and cognitive functions

The effect of compound from Example 9 on cognitive behaviors andlocomotion was assessed using active allothetic place avoidance (AAPA)task, which requires intact hippocampi and ability of animals tonavigate in space using distinct reference frames (spatial navigationand “cognitive coordination”) (Wesierska et al., 2005) and it alsoallows measuring changes in the accompanying locomotor activity. AAPAtask training involves animals trained to move over a slowly rotatinguniform circular arena, on which a prohibited sector is defined,entering which is punished by a mild footshock. A shock sector and itsposition can be determined solely by its relationship to distalorienting cues in the room (Stuchlik et al., 2008).

This task is highly dependent upon hippocampal formation, withunilateral reversible ablation of this structure with TTX leading toavoidance deficit (Cimadevilla et al., 2001). Animals solving this taskshould walk in a direction opposite to arena rotation; otherwise itwould be repeatedly brought to a fixed sector by arena rotation. Weconducted 4 acquisition session of AAPA task, during which the sectorlocation was always reinforced and remained stable throughout thetraining. Each session lasted 20 min.

Control animals obtained saline intraperitoneally (i.p.) whilstexperimental groups received subcutaneous (s.c.) injections of thecompound from Example 9 at doses 1 mg/kg and 10 mg/kg (dissolved incyclodextrin). Solution of compound from Example 9 (1 mg/2 ml or 10 mg/2ml in cyclodextrin) was applied s.c. at a volume 2 ml/kg of body weight(b.w.). Intraperitonal application of dizocilpine (a non-competitiveNMDA receptor antagonist; showing significant neuroprotective activitybut exerting cognition-disturbing and psychotomimetic effects) was usedas a positive control. Dizocilpine was applied at doses 0.1 mg/kg, 0.2mg/kg and 0.3 mg/kg. It is worth noting that dizocilpine (albeit itsexperimental neuroprotective activity) is often used to modelschizophrenia-like behaviors in humans and it has been repeatedly shownto exert a dose-dependent learning deficit and hyperlocomotion.

The results in four daily sessions are shown in FIG. 3 (means±S.E.M.).For clarity, statistical evaluations were performed for the finalsessions (representing asymptotic levels of control animals); thisapproach has repeatedly proved useful in evaluation onneuropharmacological data from AAPA task.

Locomotor activity of animals was assessed in the AAPA as total distancetraveled in the coordinate frame of the arena (without passive rotation)in each 20-min session. Animals treated with substance from example 9failed to exhibit either decrease or increase in total distance comparedto controls, whilst dizocilpine dose-dependently and consistentlystimulated the locomotor activity. The hyperlocomotion was not observedat the lowest dose of dizocilpine (0.1 mg/kg) but a marked locomotorhyperactivity was observed after 0.2 mg/kg, with an even more dramatichyperlocomotion produced by 0.3 mg/kg dose.

It should be emphasized that hyperactivity (hyperlocomotion) induced byantagonists of NMDA receptors is sometimes considered to be into certainextent analogous to human positive symptoms of schizophrenia as bothphenomena has been shown to relate to hyperfunction of mesolimbicdopaminergic circuits. Evaluation of locomotor activity in the AAPA taskhas shown that compound from Example 9 did not alter locomotor activityin this behavioral configuration. The results of activity analysis aredepicted in FIG. 3.

FIG. 3 shows the total distance per session in the AAPA training, afterapplication dizocilpine and compound from Example 9. Two highest dosesof dizocilpine caused a pronounced hyperlocomotion, whilst our compounddid not cause a significant alteration of locomotor activity. * denotessignificant difference compared to controls (p<0.05); statisticalevaluations were performed for the final session 4, which representedasymptotic level of controls.

Example 31 Number of Entrances into Shock Sector

Another parameter, which can be measured and which brings a spatialaspect to behavioral analysis, is number of entrances into prohibitedsector (occasionally termed “number of errors”). This parameter actuallyshows the overall spatial performance of the task, indicating“efficiency, in which can rats learn this task”, and it is related tocognitive functions of animals. Results have shown that neither of dosesof compound from Example 9 caused worsening in this parameter, whilstdizocilpine dose-dependently disrupted this parameter. Results of thisexperiment are shown in FIG. 4.

FIG. 4 illustrates the number of entrances (as a measure of cognitivefunctions) in every AAPA session after application of dizocilpine andcompound from Example 9. All doses of dizocilpine led to impairment inthis parameter whilst compound from Example 9 did not cause astatistical change in this parameter in comparison with controls. *denotes p<0.05 compared to controls; statistical test performed for thefinal session (see above).

Example 32 Maximum Time Between Two Errors

Another spatially selective parameter indicating cognitive abilities ismaximum time between two entrances, i.e. maximum time avoided.Dizocilpine at the three doses significantly decreased rats'performance; however, compound from Example 9 did not show this effect.Application of this compound did not significantly impaired cognitiveperformance compared to control animals. This part of experiment isgraphically illustrated in FIG. 5.

FIG. 5 shows the maximum time avoided in each session of AAPA trainingas a measure of cognitive functions after application of dizocilpine andcompound from Example 9. All doses of dizocilpine significantly impairedthis parameter, whilst compound from Example 9 did not produced anysignificant impairment. * denotes a significant difference compared tocontrols; statistical differences tested in the final session withasymptotic performance of control animals.

The following conclusion can be drawn from Examples 31-32: The resultsdemonstrate that at given dose range (1-10 mg/kg) the compound fromExample 9 does not significantly impair cognitive functions if appliedalone. In this aspect it is very different from the effects ofnon-competitive NMDA receptor antagonist dizocilpine, which causedpronounced hyperlocomotion and cognitive deficit in the place avoidancetask. These results show that compound from Example 9 at the mentioneddose range does not impair complex behavioral patterns, suggesting thatit may not disturb basal synaptic activity necessary for these functionsand could potentially be therapeutically usable if its neuroprotectiveproperties were demonstrated (see below).

Example 33 Effect of Compound from Example 9 on Spatial Cognition afterBilateral Lesion of Hippocampus by N-Methyl-D-Aspartate (NMDA)

Neuroprotective action of 3α-substituted derivatives of pregnanolone wastested in the well-established model represented by excitotoxicbilateral lesion of the hippocampus with NMDA, an agonist of NMDAreceptors. This model represents a widely-accepted paradigm ofexcitotoxicity induced experimental disruption of memory functions inlaboratory rodents. The model also mimics into some extent the excessiveaction of glutamate on cells in the hippocampus, which containsabundance of NMDA receptors. The effect of lesions and possibleneuroprotective action can be observed at various intervals aftertreatment both on the histological and behavioral level, provided that asuitable hippocampus-dependent memory task is available. In our studies,we have used a hippocampus-dependent spatial task AAPA (see above),which requires spatial and executive abilities and involves so-called“cognitive coordination” (Wesierska et al., 2005). Again, allmanipulations with animals conformed to current legislation andInstitutional recommendations on animal care and were approved byethical-care committee.

In this phase of experiment, we have studied the effect of compound fromExample 9 (and relation of this effect to dose) on NMDA excitotoxichippocampal lesion after various intervals from lesioning. 150 adultmale Long-Evans rats (250-350 g) were used in the study. Rats werehoused in transparent plastic cages (25×25×40 cm) in air-conditionedanimal room with 12/12 h dark/light rhythmicity. All rats had freeaccess to food and water throughout the study.

During surgery, animals were deeply anesthetized by isofluraneinhalation anesthesia (3.5%, driven by air). Initial sedation waspursued in initiation chamber filled with diethyl ether vapor. Duringwhole anesthesia, vital functions of animals were precisely monitored bythe person pursuing surgery. After fixation in the stereotaxic apparatus(Kopf Instruments) the skull was exposed and small trephine openingswere drilled with micro driller and subsequently, 1 ul of NMDA solution(0.09 mol NMDA in saline buffered with calcium dihydrogenphosphate topH=7.2) was microinjected into both dorsal hippocampi. The stereotaxiccoordinates (AP=4; L=±2.5; DV=4) were measured from a rigorous atlas(Paxinos and Watson, 2005) and later verified from histologicalexamination. Rats were then placed into soft post-surgery boxes.Application of compound from Example 9 took place 1) 30 min prior to and3 min after lesion, 2) 30 min after lesion, 3) 180 min after lesion, 4)24 h after lesion. Doses of compound from Example 9 were: 0.01 mg/kg,0.1 mg/kg and 10 mg/kg of b.w. In control groups and lesion-only groups,we applied only saline and NMDA, respectively. Solution of compound fromExample 9 (0.01 mg/2 ml, 0.1 mg/2 ml or 10 mg/2 ml dissolved incyclodextrin) was applied s.c. at a volume of 2 ml/kg.

Prior to behavioral observations, animals were left for 7 days torecover after surgery. After this recovery period, two days (eachconsisting of two sessions) of AAPA training were conducted. Each ratwas initially placed onto rotating (1 rpm) circular arena containing ato-be-avoided sector (60°), fixed in the coordinate frame of the room(Stuchlik et al., 2008). Upon entrance into the prohibited sector, ratwas punished by a mild electric footshock (˜0.3 mA] delivered fromlow-impedance subcutaneous wire to contact between rats paws andgrounded floor (with highest voltage drop in rats feet). Rats wore alatex harness on their backs, holding an infrared light-emitting diode(LED), which was sampled by TV camera mounted above the arena andconnected to a computer digitizing system (iTrack, Biosignal Group,USA). The shock sector is not directly perceptible for animals and itslocation can be determined solely by its relationships with distal roomlandmarks. To efficiently manage the task, animals should possessspatial learning abilities, oriented attention, and cognitivecoordination (Wesierska et al., 2005). The task is highly dependent uponhippocampus, with unilateral inactivation of this structure leading toavoidance deficit (Cimadevilla et al., 2001). This was the reason why wehave selected this task for NMDA-induced impairment and detection ofputative neuroprotective action of our compound (Example 9).

TrackAnalysis software (Biosignal Group, USA) was used to analyze theraw data. The program allows detailed analysis of animals' trajectories.Number of entrances into shock sector as well as maximum time avoidedreflects the strength of memory trace and behavioral performance and canbe used for assessment of cognitive functions.

Application of NMDA alone led to a marked hippocampal lesion and todisruption of behavioral performance in the subsequent AAPA testingcompared to control animals. Potential alleviating (and thusneuroprotective) action of our compound at various doses and aftervarious intervals from lesion are summarized in the following Examples.Graphs show means and standard errors of the mean (S.E.M.) in the finalsession, when control animals are at the asymptotic levels.

Example 34 Effect of Compound from Example 9 on the Number of Entrancesinto Shock Sector in Lesioned Animals

The compound from Example 9 at a dose 0.01 mg/kg improved the subsequentperformance in the AAPA task measured as number of entrances whenapplied at a delay of 30 min after lesion. It is interesting to pointout that if the compound was administered 30 min prior to and afterlesion, the alleviation was not significant. The compound exerted noeffect after longer intervals from the lesion. Results of theseexperiments are summarized in FIG. 6.

FIG. 6 shows the effect of compound from Example 9 at a dose 0.01 mg/kgon the number of entrances during subsequent testing in the AAPA task. Adose 0.01 mg/kg applied 30 min after lesioning ameliorated the rats'performance compared to animals with hippocampal lesion, but notreatment with our compound.

Example 35 Effect of Compound from Example 9 at a Dose 0.01 mg/kg on theMaximum Time Avoided

The compound from Example 9 had beneficial effect in lesioned animalswhen applied 30 min after lesion and also when administered at two doses30 min both prior to and after lesion with NMDA. The results are shownin FIG. 7, which illustrates the effect of our compound at a dose 0.01mg/kg on the maximum time avoided during subsequent AAPA testing.

A dose 0.01 mg/kg administered either 30 min prior to and 30 min afterlesion or 30 min after lesion improved the performance in this parametercompared to animals with NMDA lesion only.

These results suggest that the above mentioned dose of our compoundexert neuroprotective properties on the behavioral level.

Example 36 Effect of Compound from Example 9 on the Number of Entrances

A dose 0.01 mg/kg of our compound tended to exert a (non-significant)ameliorating effect on the number of errors only when applied 30 minafter lesioning, as is shown in FIG. 8. Animals applied with a dose 0.1mg/kg of our compound exhibited similar mean values as control animals;however, large variances in the measured groups likely prevented theresult from being significant. The results of this experiment areillustrated in FIG. 8, which shows the effect of our compound on thenumber of entrances into punished area during subsequent AAPA testing. Adose 0.1 mg/kg administered 30 min after lesion non-significantlyimproved the behavioral performance compared to animals with NMDA lesiononly.

Example 37 Effect of Compound from Example 9 at a Dose 0.1 mg/kg on theMaximum Time Avoided

Similar results were obtained also for maximum time avoided, as is shownin FIG. 9, which illustrates the effect of compound from Example 9 at adose 0.1 mg/kg on the maximum time avoided during subsequent training inthe AAPA task. Dose 0.1 mg/kg of our compound tended to alleviate theperformance of rats compared to lesioned animals without treatment,although this difference failed to reach statistical significance.

Example 38 Effect of Compound from Example 9 at a Dose 10 mg/kg on theNumber of Entrances in the AAPA Task

The compound from Example 9 at a dose 10 mg/kg had a significantprotective effect against hippocampal lesion only in cases, when it wasapplied at two doses (30 prior to and after lesion) and single dose (120min after lesion). No improvement was seen at other delays, as is shownin FIG. 10.

FIG. 10 illustrates the effect of compound from Example 9 at 10 mg/kg ontotal number of entrances into shock sector during subsequent AAPAlearning. A dose 10 mg/kg of our compound exerted an ameliorating effectwhen applied either 30 min prior to+after lesion or 120 min after NMDAlesion.

Example 39 Effect of Compound from Example 9 at a Dose 10 mg/kg onMaximum Time Avoided

Analysis of the maximum time avoided (another very important spatialparameter in the AAPA task) has revealed that this dose of our compoundexerted a protective effect at the same intervals as for the number ofentrances (see previous paragraph) as is shown is FIG. 11, whichillustrates the effect of compound from Example 9 at a dose 10 mg/kg onthe maximum time avoided per session in subsequent AAPA testing. A dose10 mg/kg applied 30 min prior to and after lesion and also 120 min afterlesion caused a significant improvement of rat performance compared toNMDA-lessened animals without treatment.

From previous Examples (31-37) one can infer, that compound from Example9 can, in combination with specific treatment schedules, improve thecognitive functioning of animals in comparison with animals, which weresubjected to lesion, but not to the treatment with compound from Example9. This bulk of behavioral experiments suggest a potentialneuroprotective action of compound from Example 9 on this model ofspatial learning after excitotoxic hippocampal lesion.

Example 40 Histochemical Analysis after Bilateral NMDA-InducedHippocampal Lesion

Animals were divided into four groups consisting of two series. Firstgroup of animals received an application of NMDA (intrahipocampally,i.h.) and compound from Example 9 (1 mg/kg, i.p.). Second group receivedNMDA i.h. and cyclodextrine i.p., third group obtained saline i.h. andcyclodextrin i.p. Forth group was applied with sterile saline i.h. andcompound from Example 9 at a dose 1 mg/kg, i.p. Six animals from eachgroup was sacrificed and perfused at intervals of 1-4 days afterexperimental manipulation.

Animals were deeply anesthetized by isoflurane (3.5%, driven by air) andfixed in the stereotaxic apparatus. Small drills were made in the skulland subsequently microinjection of NMDA (1 μl, 0.09 mol NMDA in saline;pH adjusted to 7.2 by calcium dihydrogenphosphate) into both hippocampiwas pursued. Coordinates of application (AP=4, L=2.5, DV=4) werelocalized with stereotaxic atlas (Paxinos and Watson, 2005) and verifiedin histological controls. Rats were again placed in soft recovery boxesafter surgery and carefully monitored after surgery.

Application of compound from Example 9 was done according toexperimental scheme either 30 min prior to and after NMDA application(in first series of experiments) or 30 min after lesion in the secondseries. Animals were perfused on days 1-4 after lesion, their brainswere fixed with paraformaldehyde and sacharose solution. The brains weresubsequently frozen to −70° C., and thin brain slices were prepared oncryomicrotome, stained and analyzed under light microscope.

After administration of NMDA, FluoroJadeB staining showed bilateraldamage of hippocampal formation within dentate gyrus, CA1-CA3, intensestaining in CA2 a dentate regions and to lesser extent in subiculum,entorhinal cortex and in prepiriform and piriform cortices. In neocortexlying within frontal and parietal lobes the cortical layers 2-5 wascytoarchitectonically altered, layer 6 was stained into lesser extent.Most prominent staining was observed in pyramidal neurons and smallinterstitial neurons of layer 4. Minor damage of basal ganglia(caudatoputamen) and thalamus in the region of reticular nucleus wasobserved. Dorsal hippocampal regions showed significantly more intensestaining than supracomissural and ventral parts of hippocampus.

After application of compound from Example 9 we have observedsignificant decrease of NMDA-induced damage to the hippocampus, mainlyin dentate gyrus and all Cornu ammonis fields and subiculum. Associatedcortical regions (entorhinal and piriform cortices) retainedhardly-quantifiable damages in archicortical layers 2 and 3 in a largeextent reaching the borders of olfactory structures. We observed apronounced cessation of damage in the whole neocortex—visual, auditoryand sensorimotor cortices. Alleviation, but not elimination of damagewas observed in caudatoputamen and thalamic nuclei. In both cases,co-localization in the confocal microscope with DAPI showed specificdamage to neurons; glial cells were spared. These results convincinglyconfirm the notion that compound from Example 9 posses a neuroprotectiveaction on the hippocampal damage by means of intrahippocampalapplication of NMDA.

Example 41 Pharmacokinetic Properties of the Compound from Example 9

In the experiment 40 adult Long-Evans male rats from breeding colony ofInstitute of Physiology AS CR v.v.i., Prague were used. Rats weretreated i.p. with a dose 1 mg/kg of compound from example 9 dissolved in72 mM of hydroxypropyl-β-cyclodextrine saline solution. Brain and plasmawas taken 5 min, 15 min, 30 min, 45 min, 60 min, 90 min, 120 min, 180min, 24 hours and 48 hours after drug administration. Each brain andblood samples were taken from separate rat. Samples were collected in 4rats for each time interval. Samples were immediately stored in a −80°C. freezer.

The concentrations of compound from example 9 in rat brain and plasmasamples were measured by high performance liquid chromatography (HPLC)coupled with mass spectrometry (Agilent 6320 Ion Trap). Deuteriedinternal standard (3_(-d) labeled compound from example 9) has been usedfor ensuring high accuracy of measurement.

Following the intraperitonal injection of compound from example 9 (1mg/kg) its c_(max)=675 ng/ml in plasma at the time of T_(max)=15 min wasdetected. The compound from example 9 penetrates blood brain barrier andrapidly enter the brain (T_(max)=60 min, c_(max)=508 ng/whole brain)after i.p injection of compound from example 9 (1 mg/kg) andexponentially decreases to a mean (4 rats in the group) of 222 ng/wholebrain after 2 hours, 128 ng/whole brain after 3 hours, 14 ng/whole brainafter 24 hours and 0.6 ng/whole brain after 48 hours. It seems that thecompound from example 9 is eliminated by a first-order process and it isnot cumulated in brain tissue. FIGS. 12 and 13 show the plasma and brainlevels of compound from example 9.

Example 42 Toxicology of the Compound from the Example 9 in Rat Model ofAcute Toxicity

Toxicity of compound from example 9 was tested on 2-month-old Long-Evansmale rats. Animals were randomly assigned to 4 groups per 7 animalseach. The first group was injected i.p. with saline, the second groupreceived i.p. cyclodextrine, the third group obtained received thecompound from example 9 at a dose 1 mg/kg (dissolved in β-cyclodextrine)and the last group compound from example 9 at a dose 100 mg/kg(dissolved in β-cyclodextrine). Animals were monitored for the period of5 days. At the end of the experiment macroscopic dissection was carriedout and organs were weighted (brain, heart, liver, spleen and kidneys).Animals' weight, food and water consumption was monitored daily. At thesame time the behavior and locomotor activity was observed and animalswere inspected for detection of effusions, constipation, etc.

There were no differences among the groups regarding in weight gain;overt behavior, locomotor activity, food and water consumption and noeffusions and constipation were detected during 5-day period. During themacroscopic dissection no differences between groups were observed andno differences were found between groups in organ weights. On the day ofapplication (15 minutes after injection) decreased locomotor activitywas registered in the fourth group (receiving i.p. 100 mg/kg of compoundfrom example 9 dissolved in cyclodextrine). Decreased locomotor activitygradually changed into anesthesia of animals (45 minutes afterinjection). Animals spontaneously awoke after 4 hours and they showed noproblems for the rest of the experiment. Anesthetic effect may berelated to the mechanism of action of this compound. FIG. 14 shows theeffect of compound from the example 9 on the body weight curve measuredover short interval. The compound was applied on day 1 at a single doseof either 1 mg/kg or 100 mg/kg.

Body weights were monitored daily and on day 5, animals were sacrificedand examined. The slight increase in β-cyclodextrine-treated animalsprobably represents between-group inhomogeneity; note that the compoundfrom example 9 at lower dose failed to affect short-term body weightgain, the higher dose tended to decrease it slightly.

FIG. 15 shows the effect of compound from example 9 on the weight ofparticular body organs. The compound was injected on day 1 and organswere weighted on autopsy on day 5. Note that there were no differencesbetween controls and animals treated with the compound from example 9.The slight decrease of liver weight in β-cyclodextrine-treated animalsprobably represents between-group inhomogeneity.

In conclusion compound from example 9 after i.p. administration in dosesup to 100 mg/kg does not exhibit any signs of acute toxicity inlaboratory rat.

Example 43 Effect of Compound from Example 9 on the Cells in Glioma-CellCulture

The effect of compound from the example 9 (the model molecule) on growthof glioma cells was studied on C6 glioma cell line (ATTC, Rockville).The model molecule was tested at doses of 0.1 and 1 μg dissolved in 50ml of β-cyclodextrine solution. Parallel control groups wereadministered by cholesterol at doses of 0.1 and 1 μg dissolved in 50 mlof β-cyclodextrine solution. The last group included in the experimentwas left intact without any application.

The cells were cultivated in vitro for 3 days (div). Afterwards testedmolecule was applied and the cell culture was studied for additional 6days. Olympus microscope with phase contrast and 200× magnification wasused for the cell monitoring. Results are shown in FIG. 16.

24 Hours after application the cells produce thinner radial projections.Later (3-6 div) the cell growth was lower compared to controls. At thesame time cells hypertrophy was observed. Faster acidification ofcultured medium implies the possibility of metabolic acceleration in thecell population. The compound from example 9 (dissolved in solution of(3-cyclodextrine) has overall slight cytostatic and pro-differentiationeffect on glioma cells studied in vitro compared to controls. The effectwas slightly apparent in control groups injected with cholesterol(dissolved in solution of (3-cyclodextrine); but it was significantlysmaller. Similar effects of compound from example 9 and cholesterol onglioma cells was due to sterane component they share, however theeffects of the compound from example 9 (dissolved in solution ofβ-cyclodextrine) are significantly amplified. These results support thepotential effect of the compound from example 1 on glioma cells in an invitro model.

Example 44 Effect of Compound from Example 9 on Anxiety Bahavior inElevated Plus Maze

The effect of i.p. administration of compound from example 9 in doses of0.001, 0.01, and 10 mg/kg on behaviour in elevated plus maze wasevaluated. The number of entrances into open arms and the total timespent in open arms were assessed.

The apparatus consisted of two opposite open arms (45 cm×10 cm) crossedat right angles with two opposite arms of the same size enclosed bywalls 40 cm high, except for the central part where the arms crossed.The whole apparatus was elevated 50 cm above the floor.

Results showed that administration of the compound from example 9exerted pronounced dose-dependent anxiolytic effect, as can be seen inFIG. 17. While tested on elevated plus maze, rats from control grouponly rarely entered open arms, thereby demonstrating some degree ofanxiety. Application of compound from example 9 had apparent anxiolyticeffect; however, only application of compound from example 9 at dose 10mg/kg led to significant increase of number of entries into open arms (*p<0.05, compared to control group).

Example 45 Effect of the Compound from Example 9 on Footshock-InducedUltrasonic Vocalization as a Model of Anxiety

Effect of compound from example 9 application at doses of 0.001 mg/kg,0.01 mg/kg and 10 mg/kg on ultrasonic vocalization after exposure tostressful situation (foot-shock) as a measure of anxiety. Underconditions of stress and pain, rats are known to emit ultrasonicdistress calls at a frequency of about 22 kHz. Under conditions ofstress and pain, rats are known to emit ultrasonic distress calls at afrequency of about 22 kHz. On the first day, rats were placed into theshock chamber individually and after 30 s of habituation, they received6 inescapable electric foot-shocks (1 shock/1 minute, 1 mA) of 10seconds duration. The procedure was repeated 24 hours later, but thistime, the animals received only one 10-second shock. Afterwards, therats were injected i.p. with vehicle (b-cyclodextrine) or the compoundfrom example 9 at doses of 0.001, 0.01, and 10 mg/kg) 30 min prior totesting. During the test session, animals were placed into the shockchamber for 10 minutes and their ultrasonic vocalization was recorded.No foot-shocks were delivered during the test session. Each animal wasrecorded separately with a Mini-3 Bat Detector (Ultra Sound Advice,London, UK). Ultrasonic vocalizations at 22-kHz consisting of calls witha minimal duration of 300 ms were recorded and analyzed by UltraVox 2.0program (Noldus, Wageningen, The Netherlands). The total number ofvocalization events and their total duration were recorded.

The results confirm slight dose-dependent anxiolytic effect the compoundfrom example 9. Significant difference is manifested only in the highestdose of 10 mg/kg, as can be seen in FIG. 18.

Example 46 Effect of the Compound from Example 9 on Prepulse Inhibitionof the Startle Response in Combination with Dizocilpine in an AnimalModel of Schizophrenia

Since the ethiopathology of schizophrenia is not fully understood, it isdifficult to establish its animal model with full construct validity.Recently proposed models are based on the central blockade of glutamatereceptors, namely their NMDA subtype. Administration of compounds whichblock NMDA receptor (MK-801, phencyclidine, ketamine) elicits apsychotic state when applied to healthy humans and worsens psychoticsymptoms when administered to schizophrenic patients. Administration ofhigh-affinity non-competitive NMDA receptor antagonist MK-801(dizocilpine) was proposed as an animal model of schizophrenia, whichproved to have relatively high predictive and phenomenological validity.Animals treated with MK-801 exhibit typical changes in behaviourincluding hyperactivity, defective habituation, impaired attention,simpler behavioural repertoire and general behavioural primitivization.Some of these symptoms can be compensated by classical and atypicalantipsychotic treatment.

Behavioural effects of antipsychotic agents have been intensivelystudied in pre-clinical models of schizophrenia-like behaviour. Majortests used are open field (locomotor activity), prepulse inhibition ofstartle response (sensorimotor gating), and learning and memoryparadigms.

The effect of i.p. administration of the compound from example 9 atdoses of 0.1 and 1 mg/kg on sensorimotor gating in prepulse inhibition(PPI) test was evaluated. All testing occurred within the startlechamber (SR-LAB, San Diego Instruments, USA), which consisted of a clearPlexiglas cylinder (8.2 cm diameter, 10×20 cm) that rested on apiezoelectric accelerometer inside a ventilated and illuminated chamber.The piezoelectric accelerometer detected and transduced motion withinthe cylinder. A high frequency loudspeaker inside the chamber (24 cmabove the animal) produced both a background noise of 62 dB and theacoustic stimuli. All rats were initially tested in a short session (5min acclimatization period plus 5 single stimuli; 120 dB strong) 2 daysbefore the experiment.

The background noise (62 dB) was presented alone for 5 min(acclimatization period) and then continued throughout the session.After the acclimatization period, the test began with five initialstartle stimuli followed by four different trial types presented in apseudorandom order: (1) single pulse: 120 dB broadband burst, 20 msduration; (2) prepulse: 13 dB above the background noise, 20 ms durationwere presented 100 ms before the onset of the pulse alone; (3) prepulsealone: 13 dB above the background noise, 20 ms duration; (4) nostimulus. A total of five presentations of each trial type were givenwith an interstimulus interval of approximately 30 s. The PPI wasmeasured as a difference between the average responses to the singlepulse and prepulse-pulse trials, and was expressed as a percent of thePPI [100−(mean response for prepulse-pulse trials/startle response forsingle pulse trials)×100]. In addition, four single pulse trials at thebeginning of the test session were not included in the calculation ofthe PPI values. We have studied the effect of MK-801 and compound fromexample 9 after combined and separate administration.

The results have shown that MK-801 disrupts PPI. Application of thecompound from example 9 to animal with induced schizophrenia-like statesignificantly dose dependently improves deficit in PPI. Application ofcompound from example 9 alone has shown no effect on sensorimotor gatingmeasured in this paradigm (see FIG. 19). Prepulse inhibition wasconsiderably disrupted after MK-801 0.1 mg/kg treatment (** p<0.01,compared to control group). However, co-administration with eithercompound from example 9 at dose 0.1 mg/kg or 1 mg/kg blocked theexacerbating effect of MK-801. Application of compound from example 9alone had no influence on prepulse inhibition.

Example 47 Effect of the Compound from Example 9 Alone or in Combinationwith Mk-801 on the Locomotion in the Open-Field Behavior in an AnimalModel of Schizophrenia

The effect of the compound from example 9 in doses of 0.1 mg/kg and 1mg/kg on locomotor activity in open field test was evaluated. Locomotoractivity expressed as a total distance travelled during 30 minexploration of a box (68×68×30 cm) located in a sound proof room wasmeasured using a video tracking system for automation of the behaviouralexperiments (Noldus, EthoVision, Version 2.1.).

The results have shown that contrarily to application of MK-801,administration of the compound from Example 9 did not produce locomotoralterations in the open-field test. However application of the compoundfrom example 9 to animal with induced schizophrenia-like state has noteffect on hyperlocomotion state induced by MK-801. To sum up thecompound from example 9 has pro-cognitive effect without affecting oflocomotor alterations (FIG. 20).

Example 48 Effect of the Compound from Example 9 on Behavior of Rats inthe Active Allothetic Place Avoidance Task After Administration ofMK-801 as an Animal Model of Schizophrenia (Locomotion)

The effect of compound from example 9 application on changes in behaviorand locomotor activity in animal model of schizophrenia was monitored.Schizophrenia-like behavior in rats was induced by dizocilpine (MK-801)administration. Dizocilpine was administered in doses of 0.001 mg/kg,0.01 mg/kg, 0.1 mg/kg and 10 mg/kg. Spatial orientation and cognitivefunction of rats were assessed in active allothetic place avoidance task(AAPA).

Schizophrenia-like behaviour in rats was induced by administration ofdizocilpine (MK-801). Spatial navigation and cognitive functions of ratswere assessed in active alothetic place avoidance task (AAPA). Inaddition, this task allows simultaneous assessment of potentialdifferences in locomotory activity. The AAPA task requires rats movingon a continuously rotating (1 rpm) smooth metallic circular arena toavoid an unmarked place defined in the coordinates of the experimentalroom. The avoidance is reinforced by a mild electric foot-shockadministered automatically upon entering the to-be avoided place.

The AAPA task is highly dependent on hippocampal function, since even aunilateral inactivation of thstructure hippocampus impairs thisbehaviour. Animals have to actively and repeatedly move away from theto-be-avoided place otherwise they are brought there by the arenarotation. Each AAPA session lasts 20 min and 4 sessions were conductedover 4 consecutive days.

Rats received 0.1 mg/kg MK-801 alone or co-administered with thecompound from example 9 (dissolved in β-cyclodextrine) in doses of 0.001mg/kg, 0.01 mg/kg, 0.1 mg/kg, and 10 mg/kg. Control animals receivedsaline. While MK-801 shows neuroprotective effect in some experiments,it also produces cognitive deficits and hyperlocomotion in a stateanalogous to psychosis. It is for a common animal model ofschizophrenia-like behaviour.

The results are shown in FIGS. 21-23, where means and standard errors ofthe mean (S.E.M.) are represented for individual sessions. Statisticanalysis was for better clarity stated always for the last session, whenan asymptotic level of performance in control animals was reached.

Locomotion activity in AAPA task was evaluated as a total distancetravelled in the arena during 20-minute session. Animals that were givencompound from example 9 at does of 0.001 mg/kg, 0.01 mg/kg and 0.1 mg/kgshowed neither increase nor decrease of locomotion activity in any ofthe doses used in comparison to control rats. The only exception was thedose of 10 mg/kg that increased locomotor activity compared to controls.Whereas the dose of 0.1 mg/kg MK-801 did not lead to enhancedlocomotion, it is well known that higher doses (from 0.2 mg/kg)considerably increase locomotion.

In literature hyperlocomotion is often considered to be an experimentalanalogy of positive symptoms of psychosis because it is related toexcessive functioning of dopamine system in mesolimbic brain regions.Evaluation of locomotion activity in AAPA task showed that compound fromexample 9 does not change this activity in all doses except that of 10mg/kg. Results of locomotion activity measurements are pictured at theFIG. 21. Only the highest dose of the compound from Example 9 increasedlocomotor activity in combination with 0.1 mg/kg dizocilpine. Thissuggests that compound affect behavior only at highest dose incombination with psychotomimetic NMDA blocker.

Example 49 Effect of the Compound from Example 9 on the Number of Errorsin the Aapa Task in Combination with MK-801 in an Animal Model ofSchizophrenia

The main measure of the avoidance behaviour is the number of errors(entrances into the to-be-avoided place. Administration of MK-801 (0.1mg/kg) resulted in marked impairment of this parameter.Co-administration of the compound from example 9 reversed this deficitsat all doses. The results are summarized in FIG. 22.

Graph shows the number of errors (entrances into restricted sector) as ameasure of cognitive functions for every session in AAPA task afterapplication of saline, dizocilpine 0.1 mg/kg alone or in co-applicationwith compound from example 9. All of compound from example 9 dosesresulted in improvement of cognitive deficits induced by dizocilpineadministration.

Example 50 Effect of the Compound from Example 9 on Maximum Time ofAvoidance in the AAPA Task in Combination with MK-801

The maximum latency between two errors is another measure of theavoidance. MK-801 (0.1 mg/kg) produced a significant deficit in thisparameter. Co-administration of the compound from example 9 reversedthis deficit in doses of 0.001, 0.01, and 10 mg/kg. The results aresummarized in FIG. 23.

Graphs show the maximum time of avoidance as a measure of cognitivefunctions for every session in AAPA task after application of saline,MK-801 alone or in co-application with compound from example 9. MK-801application resulted in worsening of this parameter; on the other handapplication of compound from example 9 at doses of 0.001 mg/kg, 0.01mg/kg, 0.1 mg/kg and 10 mg/kg led to statistically significant change ofcognitive functions.

In conclusion, the results from examples 21-23 indicate that thecompound from example 9 reverses the cognitive deficit induced byMK-801. Hence, the compound from example 9 can be used to alleviate thecognitive deficit in an animal model of schizophrenia.

Example 51 Effect of the Compound from Example 9 on the LearnedHelplessness in a Model of Affective Disorders

Learned helplessness (LH) has been associated with several differentpsychological disorders. Depression, anxiety, phobias, shyness andloneliness can all be exacerbated by learned helplessness. Learnedhelplessness occurs when a subject is repeatedly subjected to aninescapable aversive stimulus. Eventually, animals will stop trying toavoid the stimulus and behave as utterly unable to change the situation.Even if opportunities to escape open, the learned helplessness preventsany action.

Male Long-Evans rats weighing 350-450 g were used. Animals were housedin groups in an air-conditioned room with 12 h/12 h light/dark cyclewith the lights on at 7 a.m. Water and food were available ad libitum.Animals were randomly assigned to 3 groups—control group (received noshocks), learned helplessness group (received shocks) and experimentalgroup (received shocks and injection of compound from example 9). Anexperimental set-up was composed of two chambers separated by a slidingdoor. The transparent shock chamber (48×20×20 cm) with one entrance ledto the dark chamber (25×15×15 cm) made of black plexiglass. The floor ofthe shock chamber consisted of metal rods. Shocks were delivered throughthe grid floor. A shock generator (Data acquisition MF624 multifunctionI/O board for PCI, Humusoft Ltd., Czech Republic) was connected to andcontrolled by a computer. On the pre-treatment day rats wereindividually placed into the apparatus for 10-minute habituation period.The sliding door separating two chambers was removed allowing freemovements between the compartments. Immediately after the habituationrats were confined to the shock chamber for 1 hour. Inescapable shocks(72 V) of 10 s duration were delivered at 50 s intervals. Afterwardsanimals were injected i.p. with compound from example 9 in dose of 1mg/kg. Control animals received no shocks during 1 hour shock sessionand animals from learned helplessness group received no drug. The nextday, 24 hours later, each animal underwent escapable shock trials. Thenumber of escapes was recorded. An escape failure referred to failedcrossing response to the adjacent dark chamber during the shockdelivery.

The application of the compound from example 9 significantly increasednumber of escapes. These results strongly support the potentialantidepressant action of compound from example 9 (see FIG. 24).Experienced learned helplessness (LH) had a great impact on escapesperformed in the LH apparatus. It considerably reduced number ofattempts to escape (*** p<0.001, compared to control group). On thecontrary, application of compound from example 9 fully blocked thedebilitating effect of previous learned helplessness experience (##p<0.01, compared to LH group).

Example 52 Effect of the Compound from Example 9 on Forced Swim TestUsed as the Experimental Model of Depression and Influence of ExcessiveStress

Forced swim test is an experimental model of depression and excessivestress used in laboratory rodents. Rats were randomly assigned to twogroups of seven animals each. Control group received an injection ofcyclodextrine (i.p.) 60 min. prior to experiment, while experimentalgroup was injected i.p. with the compound from example 9 in a dose of 1mg/kg 60 minutes prior to the experiment. Rats were forced to swim bybeing individually placed into a bucket (30 cm diameter, 28 cm height)containing water (25° C.). Immobility (floating) in the water isinterpreted as a state of despair when the animals realize that anescape is impossible. Immobility was defined as floating (as opposed toactive swimming) with minimal effort required for keeping the head abovewater. All animals received a single 30-minute trial and their behaviourwas monitored by an experimenter. The trial was divided into six5-minute intervals and floating of at least 30 second duration withineach interval was recorded by the experimenter.

In conclusion, treatment of the compound from example 9 resulted inextended time of swimming. Rats from group treated by compound fromexample 9 (1 mg/kg) spent significantly less time by floating, incomparison to controls (p<0.001), as is apparent from longer averagetime to reach immobility (FIG. 25). Moreover in experimental grouptrials (all but one) needed not to be forcibly terminated due to animalssinking. The compound from example 9 exhibits antidepressant propertiesin forced swim test used as an animal model of depression.

Example 53 Effect of the Compound from Example 9 on Depression-LikeBehavior Induced by Social Defeat

Glutamatergic neurotransmission plays an important role in stressresponse and development of mood disorders, depression, and otherstress-related disorders. The aim of this experiment is to verify thehypothesis that treatment with the compound from example 9 affectsbehavioural response to chronic stress. A mouse model of repeated socialdefeat (nonaggressive male mice repeatedly defeated by aggressivecounterparts) was used.

Naive adult male mice (albino out-bred strain ICR, VELAZ s.r.o., Prague,Czech Republic, 30-37 g) were used in this study. Food and water wereavailable ad libitum. Mice were housed either individually without anyhandling in self-cleaning cages with a grid floor (8×6×13 cm) or ingroups of 17-20 in standard plastic cages (38×22×14 cm) with the floorscovered with wooden shavings. After 3 weeks, each individually housedmouse was allowed 30 min adaptation in a Plexiglas neutral observationcage (20×20×30 cm) with clean wooden shavings before it was coupled witha group-housed male partner for 4 min/day. As experimental subjects inExperiments 1 and 2 served the group-housed mice with or without defeatin four conflict dyadic interactions, the singly housed mice exhibitingaggressive behavioural activities were received 1 week apart. Numbers ofattacks, tail rattling and threats exhibited by singly housed mice wererecorded and evaluated by the hardware/software Observer 3.1, NoldusTechnology, Holland. Animals were housed, and behavioural testing wasperformed in a different room during the light phase of the constantlight-dark cycle with lights on at 6:00 and off at 18:00 h. Temperaturewas maintained at 21° C., and relative humidity was 50%. The mice werenot handled except on the experimental days.

Group-housed males were used for the experiment. In the first part ofthe experiment, mice were given β-cyclodextrine or compound from example9 at doses 1 and 10 mg/kg orally via a syringe, in a randomized order,30 min prior to the open field observations performed in the identicalanimal house but a different room from that used for the social defeatinteractions. Each animal was placed singly into the centre of a novelenvironment (arena 30×30 cm) of the PC controlled tracking apparatusActi-track (Panlab, S. L., Spain) with infrared beam sensors. Over a3-min testing period, the distance traveled, as a marker oflocomotor/exploratory behaviour, in the open field was measured. Twodays later, each mouse was defeated with a singly housed mouseexhibiting aggressive behaviour in a 4-min agonistic interaction. Theprocedure was repeated four times, 7 days apart. Immediately after thelast (fourth) agonistic interaction, each mouse was administered thesame treatment as in the first part of the experiment. Thirty minutesfollowing the drug administration, the animal was placed into the openfield arena and distance traveled was measured, as described before.Data of two group-housed mice, which were seriously wounded during theaggressive dyadic encounters, were excluded from the statisticalanalysis.

Acute treatment of mice with compound from example 9 at doses 1 and 10mg/kg reduced a hypolocomotion in an open field induced by agonisticinteractions in mice. The results demonstrate that compound from example9 can disinhibit suppressed locomotor activity in repeatedly defeatedgroup-housed mice. This strongly supports the beneficiality of thecompound from Example 9 on diseases involving stress-related behavioralterations.

In conclusion, presented effects of compound from example 9 observed inthe social stress model used, which is close to real life events andemulates human psychiatric disorders, suggest possibleantidepressant-like properties of the compound. See illustrative FIG.26. In control mice group, repeated social interaction with aggressiveconspecific yielded low locomotion in open-field test. Application ofthe compound from example 9, however, unblocked the suppressive effectof defeat experience and significantly increased locomotion inopen-field.

Example 54 Effect of the Compound from Example 9 on the Pain Phenomena

The effect of the compound from example 9 was assessed on acutenociception in rat. Pain was evaluated by measurement of paw/tailwithdrawal latencies. Male Wistar rats (Velaz, Czech Republic) wereused. Animals were kept at 22° C. (relative humidity was 40-70%) under a12-hour light/12-hour dark cycle. All experiments were approved by theCommittee for Animal Care and conducted in accordance with the ethicalguidelines of the International Association for the Study of Pain. Theanimals were randomly divided into experimental and testing groupsgroups. The compound from example 9 was dissolved in β-cyclodextrine(the concentration 1.0 mg/ml and 10 mg/kg injected intraperitoneally or(3-cyclodextrine injected intraperitoneally in same volume).

Thermal nociception was determined using plantar test equipment (UgoBasile, Italy). The latency (in seconds) of forelimb, hindlimb or tailwithdrawals to the noxious thermal stimulation was measured. Each animalwas placed individually in a clear plastic box with a clear glass floor,and was allowed to acclimatize for 10 min; the temperature and humidityin the testing room were kept in the same range as those in the housingrooms. The latencies were measured 3 times per session, separated by aminimum interval of 5 min, and the mean value was used for furtheranalysis. Infrared intensity was set to 40% and the maximum cutoff valuewas set at 22 seconds to avoid thermal injury. The testing box wascleaned after each session.

The compound from Example 9 in dose 10 mg/kg applied to tail suppressedreactivity to painful stimuli and significantly prolonged interval ofresistance to a painful stimulus, while the lower dose of this compounddid not yield such an effect. These results strongly support the conceptthat the compound from example 9 exerts moderate anti-nociceptive andanalgesic effects in animal model. Results of this experiment aresummarized in the FIGS. 27 and 28.

After application of the compound from example 9 at 10 mg/kg dose, ratsexpressed significantly prolonged interval of resistance to a painfulstimulus aimed at all their four limbs (*** p<0.001, compared to controlgroup “after”; ### p<0.001 compared to compound from example 9 in dose10 mg/kg “before”). Such an effect was not however observed afterapplication of compound from example 9 at 1 mg/kg dose (FIG. 27). Inanother experiment higher dose of compound from example 9 also exertedan antinociceptive effect in pain-induced tail withdrawal (data notshown).

Likewise, the compound from example 9 at 10 mg/kg dose applied to tailsuppressed reactivity to painful stimuli; while the lower dose of thecompound from example 9 did not yield such an effect, see FIG. 28.Beneficial action of the compound from example 9 on the neuropathic painmodel has also been documented by our experiments. These resultsstrongly support the concept of anti-nociceptive action of compound fromexample 9 in an animal model.

Example 55 Effect of the Compound from Example 9 on the Ischemic Damageof the Neural Tissue

The effect of compound from example 9 on cognitive coordination andmotor activity was examined in stroke model of ischemic brain injury.Ischemic-hypoxic damage was induced by bilateral occlusion of commoncarotid arteries and subsequent confinement in box with 10% O₂ (for 30min). The animals were injected i.p. by compound from example 9dissolved in β-cyclodextrine (1 mg/kg) immediately after their removalfrom box. Ischemic group was injected only by β-cyclodextrine in thesame time and volume. Surgery was conducted under anesthesia (xylazine 6mg/kg, thiopental 50 mg/kg).

It is known that ischemia-hypoxia causes neuronal necrosis inspecifically vulnerable areas of brain. The two-vessel occlusion/hypoxicmodel gives rise to stroke changes, for example in cells of thehippocampus, caudatoputamen and neocortex. As such structures areintimately involved in memory, cognitive and motor processes,ischemia/hypoxia leads to impairment in these functions. Used animalmodel of stroke damage dealing with ischemia/hypoxia has enabled theidentification of deleterious phenomena that may be corrected orneutralized by drugs.

The effect of ischemic was evaluated a week after surgical procedures inAAPA task, beam walking and rotarod. Ischemia led to deterioration ofmotor activity (rotarod and beam walking) and deficit in cognitivecoordination. Interestingly, total path in AAPA was not decreased.

Application of the compound from example 9 has no effect on disturbanceof motor changes in rotarod and beam walking test (data not shown). Butit improves cognitive deficit induced by ischemic/hypoxic procedures inAAPA task. (See FIG. 29). As a conclusion the compound from example 9significantly counteracted cognitive deficit induced by ischemic hypoxicdamage but without any effect on motor changes in dose of 1 mg/kg.

Ischemic rats treated with the compound from example 9 however performedequally well as controls (## p<0.01 compared to ischemic rats)demonstrating strong protective effect of the compound. None of theexperimental condition affected locomotion in AAPA. All groups reachedsimilar level of elapsed path during a session.

Example 56 Effect of the Compound from Example 9 on the Animal Model ofAlzheimer Disease and Age-Related Cognitive Decline

The effect of the compound from example 9 on an animal model ofage-related cognitive decline and neurodegenerative disorders (includingAlzheimer disease) is documented. In the present experiment the effectof central muscarinic blockade by systemic administration of scopolamineat dose 2.0 mg/kg on both reinforced retention of the AAPA andre-acquisition of the AAPA in a new environment was investigated (by thestandard method described previously; see Vales and Stuchlik, 2005).

The compound from example 9 was dissolved in f3-cyclodextrine (at aconcentration of 1.0 mg/ml). Animals were injected intraperitoneallywith 1 ml/kg b.w. of this solution or with 1 ml/kg b.w. ofβ-cyclodextrine. All animals thus received the same volume of liquid perbody weight in each injection. Injections were delivered 40 min prior tobehavioural testing). Scopolamine hydrobromide (scopolamine; SigmaAldrich) was dissolved in saline (0.9% NaCl, 2.0 mg/ml). Animals wereinjected intraperitoneally with 1 ml/kg b.w. of this solution or with 1ml/kg b.w. of saline. All animals thus received the same volume ofliquid per body weight in each injection. Injections were delivered 20min prior to behavioural testing. Adult male hooded rats of theLong-Evans strain (3 months old; weighing 300-400g) was used.

The active allothetic place avoidance apparatus is described in example28. Initially, non-injected rats were trained to avoid a sector locatedin the north of the Room 1 for four consecutive daily sessions (sessions1-4). Subsequently, in session 5 they were injected with ether saline orscopolamine at the above-mentioned doses and the effect of the drug onwell-pretrained place avoidance was examined (reinforced retention).During the following weekend, rats were left in the animal room withoutany manipulation. The next week, rats were trained under saline orscopolamine in the AAPA in the novel Room 2 for 5 days (sessions 6-10)in order to test re-acquisition of AAPA in a new environment, when theywere already familiar with the procedural component of the AAPA task.The shock sector was defined in the south of the room in the Room 2arena.

The rats were randomly divided into experimental groups. Group CONTROLS(n=8) received intraperitoneal injections of saline, second group(termed “scopolamine” in the FIG. 30) (n=8) received intraperitonealinjections of scopolamine at the dose of 2.0 mg/kg, and third group(n=8) received intraperitoneal injections of scopolamine at the dose of2.0 mg/kg and compound from example 9 at the dose of 1.0 mg/ml i.p.respectively. Scopolamine and compound from example 9 were administeredon days 5-9. Initial training without treatment was performed in arena 1(sessions 1-5; with session 5 as reinforced retrieval under the effectof compounds) and reacquisition on a new arena was tested on days 6-9.)Note that the compound from example 9 tended to partly alleviatingscopolamine induced deficit on days 5-9.

Effects of the compound from example 9 are documented here on the numberof entrances, a spatially-selective parameter, which used frequently inthe place avoidance studies, see FIG. 30. Visual inspection (during andafter the injection) did not reveal any vocalizations, increaseddefecation, ataxia, or any other signs of behavioral discomfort. Ratswere able to maintain the correct postural positions and their mainneurological reflexes were preserved (grasping reflex, tilted platformtest, contact placing reactions).

The results suggest that animals treated with the 2.0 mg/kg dose ofscopolamine exhibited a significant impairment of place avoidanceperformance, both in the reinforced retention session, andre-acquisition training. This deficit tends to be decreased by i.p.administration of the compound from example 9. These results support theconcept of protective effect of the compound from example 9 onage-related cognitive decline, Alzheimer dementia and possible otherneurodegenerative disorders.

Example 57 Effect of Compound from Example 9 on the SpontaneousElectroencephalogram (EEG) and Evoked Potentials in Awaken Rats

To assess the influence of the compound from example 9 on spontaneousEEG, evoked potentials and epileptic afterdischarges, recordingelectrodes were implanted into the epidural spaces of the adult Wistarrats. Rats were anaesthetized with isoflurane (1.5-2%) and 4 silver wirebare electrodes were inserted epidurally over sensorimotor cortex.Reference and ground electrodes were placed above cerebellum. The wholemontage was fixed to the skull with dental acrylic.

After recovery period (1 week), animals were placed into the plasticboxes and connected to the Video EEG monitoring (VisionBrain/). After 1hour of background recording the compound from example 9 wasadministered at the dose of 0.01 mg/kg and EEG was recorded for next 24hours. Analysis of EEG was performed in a custom-based MATLAB script.Power of the EEG signal was calculated from FFT in frequency band 1.5-45Hz. Length and duration of spike and wave afterdischarges (SPW) wasmeasured from EEG to assess effect of compound from example 9 (0.01mg/kg and 10 mg/kg) on epileptiform activity.

FIG. 31 shows the effect of compound from example 9 on the epilepticafterdischarges elicited by stimulation of the rat somatosensory areas.Note that the compound from example 9 tended to slightly decrease bothduration and number of spike-wave episodes, although higher dose tendedto increase these parameter (although the study requires furtherextension using more experimental subjects). This suggests a biphasicaction of the compound from example 9 of the animal model ofexperimentally-induced epileptiform activity (SPW spike and wave).

Effect of the compound from example 9 on the spontaneous EEG power wasmeasured at intervals 60 min and 180 min after application; see FIG. 32.Note that the compound from sample 9 changes the EEG power at theshorter interval.

The results strongly support the effect of compound from example 9 onthe spontaneous EEG activity. Changes in power accompany variousphysiological (sleep) and pathophysiological conditions. Effect of thecompound from example 9 on epileptic afterdischarges appears to bebiphasic, with low doses tending to decrease this type of seizures,whilst higher doses may exacerbate it.

INDUSTRIAL APPLICABILITY

The compounds mentioned in the presented patent are industriallyproducible and applicable for treatment of numerous diseases of centralnervous system, e.g. the following:

1) hypoxic and ischaemic damage of the central nervous system, stroke,and other excitotoxicity-induced pathological alterations.2) neurodegenerative changes and disorders3) affective disorders, depression, PTSD and other stress-relateddiseases4) schizophrenia and other psychotic disorders5) pain, hyperalgesia and disorders of nociception6) addictive disorders7) multiple sclerosis and other autoimmune diseases8) epilepsy and other seizure disorders9) hyperplasic changes in CNS, CNS tumours including gliomas

LITERATURE CITED

-   Cimadevilla J M, Wesierska M, Fenton A A, Bures J; Inactivating one    hippocampus impairs avoidance of a stable room-defined place during    dissociation of arena cues from room cues by rotation of the arena.    Proc. Natl. Acad. Sci. USA 98 (2001), 3531-3536.-   Morrow A L; Recent developments in the significance and therapeutic    relevance of neuroactive steroids-Introduction to the special issue.    Pharmacol Ther. 2007, 116(1), 1-6.-   Paxinos G, Watson C R; The Rat Brain in Stereotaxic Coordinates (5th    ed.), Elsevier Academic Press, San Diego (2005).-   Petrovic M, Sedlacek M, Horak M, Chodounska H, Vyklický, L Jr.;    20-oxo-5beta-pregnan-3alpha-yl sulfate is a use-dependent NMDA    receptor inhibitor. J. Neurosci. 2005, 25(37), 8439-50.-   Stuchlik A, Petrasek T, Vales K; Dopamine D2 receptors and alpha    1-adrenoceptors synergistically modulate locomotion and behavior of    rats in a place avoidance task. Behay. Brain Res. 189 (2008),    139-144.-   Vales K, Stuchlik A; Central muscarinic blockade interferes with    retrieval and reacquisition of active allothetic place avoidance    despite spatial pretraining. Behav Brain Res. 161 (2005), 238-44.-   Villmann C, Becker C M; On the hypes and falls in neuroprotection:    targeting the NMDA receptor. Neuroscientist. 2007, 13(6), 594-615.-   Weaver C E, Land M B, Purdy R H, Ruchards K G, Gibbs T T, Farb D    H; J. Pharm. Exp. Ther. 293 (2000), 747.-   Wesierska M, Dockery C, Fenton A A; Beyond memory, navigation, and    inhibition: behavioral evidence for hippocampus-dependent cognitive    coordination in the rat. J. Neurosci. 25 (2005), 2413-2419.

1. Compounds of general formula I

where R¹ stands for group with general formula R³OOC—R²—C(R⁴)—R⁵—, whereR² stands for alkyl or alkenyl group with 1 to 18 carbon in a straightor a branching carbon chain, which may be substituted by one or morehalogen atoms and amino group, which may be either free or protected bya removable protecting group, alkoxycarbonyl group, aromatic group,and/or heterocyclic group, in which the heteroatom means oxygen atom,sulfur, or nitrogen atom; R³ represents either a hydrogen atom or aprotecting group of carboxyl groups, preferably benzyl group; R⁴represents oxygen atom, nitrogen atom or a sulfur atom bound by a doublebond or R⁴ represents two hydrogen atoms; R⁵ represents any at leastbivalent atom, preferably an oxygen atom, the nitrogen, or carbon atom,except when R² represents the group (CH₂), where n=0-3 andsimultaneously R³ represents a hydrogen atom and R⁴ and R⁵ represents anoxygen atom.
 2. Method of production of compounds of general formula Iaccording to claim 1, characterized in that3alfa-hydroxy-5beta-pregnan-20-one (formula II)

is transferred to a compound of general formula I, where R¹ means thesame as given above and R⁴ represents oxygen atom so that the particulardicarboxylic acid, dicarboxylic acid with protected amino group or,where applicable, dicarboxylic acid protected on a one carboxylic groupis dissolved in a suitable solvent that allows to remove the remainingwater, preferably in benzene or toluene, most preferably in benzene,whereupon after the removal of water by a partial distillation of thesolvent the reaction mixture, which is prevented against the watersupply in an appropriate manner known in the art, is cooled down to roomtemperature and under the inert atmosphere is slowly added condensingagent, preferably DCC, and a solution of compound formula II in asuitable solvent, preferably in an aromatic hydrocarbon, advantageouslyin benzene or toluene, most preferably in benzene, in the presence of acatalytic agent, preferably DMAP; the reaction mixture is then stirred10-48 hours, preferably overnight, at temperatures from 0 to 50° C.,preferably at room temperature and the next day the mixture is pouredinto saturated sodium bicarbonate, preferably aqueous NaHCO₃ or KHCO₃,and the product is extracted with an organic solvent, in which is wellsoluble, for example and advantageously, ethyl acetate; collectedorganic phases are washed with water to remove sodium bicarbonate,precipitated N,N′-dicyclohexylurea is filtered off and the filtrate isdried over drying agent, preferably magnesium sulfate or sodium sulfate,most preferably sodium sulfate and the solvent evaporated, preferablyunder vakuum; the obtained product is purified, where appropriate,preferably by a chromatography on a column of silica gel to afford thecompound of general formula I, where R¹ represents the group of thegeneral formula R³OOC—R²—CO— and R² represents alkyl or alkenyl groupwith 1 to 18 carbon atoms in a straight or a branching carbon chain,which may be substituted by one or several halogen atoms and by aminogroup, which is protected by a group that allows deprotection; R³represents a protecting group for carboxyl groups, preferably benzylegroup that can be removed so that the obtained compound is dissolved ina suitable solvent, preferably alcohol, most preferably in methanol, andto this solution a hydrogenation catalyst is added, preferably Pd/CaCO₃;after the hydrogenation, the catalyst is filtered off and the solvent isevaporated to afford the product of general formula I in which R¹represents the group of general formula R³OOC—R²—CO—, where R² meansalkyl or alkenyl group with 1 to 18 carbon atoms in a straight or abranching carbon chain, which may be substituted by one or severalhalogen atom and amino group, which is protected by a group that allowsdeprotection and R³ represents a hydrogen atom.
 3. Method of productionof compounds of general formula I according to claim 2, characterized inthat R¹ represents the group of general formula R³OOC—R²—CO—, where R²represents alkyl or alkenyl group with 1 to 18 carbon atoms in astraight or a branching carbon chain which may be substituted by one ormore halogen atoms and amino group, which is protected by a removablegroup and R³ represents a hydrogen atom and has an amino group, which isprotected by removable group; the deprotection of amino group isaccomplished so that the compound is dissolved in an organic solvent,preferably in methylen chloride and trifluoroacetic acid is addend, thenthe reaction mixture is allowed to react from 0.1 to 48 hours,preferably 16 hours at temperatures from 0° to 50° C., preferably atroom temperature; when the solvent is removed, the residue is dissolvedin an organic solvent, preferably in methanol, then pyridine is addedand the mixture is evaporated to dryness to obtain the product ofgeneral formula I, where R¹ represents the group of general formulaR³OOC—R²— CO—, where R² means alkyl or alkenyl group with 1 to 18 carbonatoms in a straight or a branching carbon chain, which may besubstituted by one or more halogen atoms and amino group and R³represents a hydrogen atom.
 4. Method of production of compounds ofgeneral formula I according to claim 1, characterized by that when R² ofcompound of general formula I contains a heterocyclic group, as forexample in the compound of formula HET

such heterocyclic group is introduced into a molecule of general formulaI for example by the reaction of activated carboxyl group with aminosubstituent on the alkyl chain R² whereas this carboxyl group may befunctionalized with an activating group, such as hydroxybenzotriazole,substituted hydroxybenzotriazole, HATU group, TATU group and with theadvantage TSTU group in the form of succinimidylester, where this esterreacts with the compound of general formula I in which R² means alkylgroup substituted with amino group so the compound of general formula Iis obtained, in which R² represents alkyl group substituted with aminogroup that is substituted with the heterocyclic group of HET.
 5. The useof the compounds with general formula I according to claim 1 forproduction of pharmaceuticals treating various diseases of centralnervous systems, e.g. such as the following a) ischemic damage of CNS,b) neurodegenerative changes and disorders, c) affective disorders,depression, PTSD and diseases related to stress, d) schizophrenia, e)pain, f) addictions, g) multiple sclerosis, h) epilepsy, i) gliomas. 6.The use of the compounds with general formula I according to claim 1 intreatment of neuropsychiatric disorders related to imbalance ofglutamatergic neurotransmitter system, ischemic damage of CNS,neurodegenerative changes and disorders of CNS, affective disorders,depression, PTSD and other diseases related to stress, anxiety,schizophrenia and psychotic disorders, pain, addictions, multiplesclerosis, epilepsy and gliomas.
 7. The use of the compounds withgeneral formula I according to claim 1 for production of veterinary andhuman pharmaceutical preparations used in treatment of neuropsychiatricdisorders related to imbalance of glutamatergic neurotransmitter system,ischemic damage of CNS, neurodegenerative changes and disorders of CNS,affective disorders, depression, PTSD and other diseases related tostress, anxiety, schizophrenia and psychotic disorders, pain,addictions, multiple sclerosis, epilepsy and gliomas.
 8. The use of thecompounds with general formula I according to claim 1 for production ofsubstances utilized in experimental research, analytic chemistry,dietary supplements or cosmetic preparations.
 9. The use according toclaim 5, where compounds with general formula I according to claim 1 aredissolved in β-cyclodextrine.